Application of shRNA for inhibiting BCL-9 and recombinant virus vector in treatment of retinal angiogenesis related diseases

Abstract

The invention provides application of shRNA (short hairpin Ribonucleic Acid) for inhibiting BCL-9 and a recombinant virus vector to treatment of retinal angiogenesis related diseases. According to the present invention, the shRNA capable of effectively inhibiting BCL-9 gene expression in the targeting manner is obtained through screening, and the recombinant virus vector containing the shRNA is constructed; after the shRNA or the recombinant virus vector is introduced into the human retinal vascular endothelial cells in an injection mode to specifically inhibit the expression of the BCL-9 gene, the activity, the multiplication capacity and the tube forming capacity of the human retinal vascular endothelial cells can be effectively reduced, and retinal angiogenesis is inhibited. Therefore, the shRNA and the recombinant virus vector for specifically inhibiting the expression of the BCL-9 gene can be used for treating retinal angiogenesis related diseases, including diabetic retinopathy, neovascular age-related retinopathy, premature infant retinopathy and the like. The invention provides a new target for treatment of retinal angiogenesis related diseases and develops a new idea.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of shRNA and recombinant virus vectors that inhibit BCL-9 in treating retinal angiogenesis-related diseases. Background Art

[0002] Angiogenesis refers to the formation of new blood vessels from existing capillaries or post-capillary veins. The main processes include: degradation of vascular basement membrane during the activation period, activation, proliferation, migration and reconstruction of endothelial cells to form new blood vessels and vascular networks. It is a complex process involving multiple cells and molecules, and the mechanisms regulating angiogenesis vary greatly in different organs and diseases.

[0003] Retinal neovascularization is an important pathological feature of retinal fundus diseases and is associated with a variety of retinal diseases, such as diabetic retinopathy, neovascular age-related retinopathy, and retinopathy of prematurity. The generation of retinal neovascularization is an adaptive biological response to retinal ischemia and hypoxia, and hyperglycemia can also damage the retinal vascular endothelium, leading to the destruction of the integrity of the blood-retinal barrier (BRB), increased permeability of retinal microvessels, and subsequent neovascularization. However, retinal neovascularization caused by ischemia, hypoxia, and hyperglycemia cannot function like normal blood vessels. The permeability of the vascular wall of the new blood vessels is high, which may cause subretinal hemorrhage, lipid exudation, detachment of the retinal pigment epithelium and choroid, and the formation of fibrotic scars, resulting in irreversible damage to vision.

[0004] The generation of retinal neovascularization is a complex process, in which different pathways are involved. Ultimately, it will affect the proliferation, migration, adhesion and differentiation of vascular cells through vascular endothelial growth factor (VEGF), vascular cell adhesion molecule-1 (VCAM-1), angiopoietin (ANGPT), intercellular adhesion molecule 1 (ICAM-1) and other factors related to vascular development, generation and leakage, thus causing retinal neovascularization and vascular leakage. How to effectively control the generation of retinal neovascularization and prevent its occurrence in time is the key to treating many retinal diseases.

[0005] Currently, there are limited effective clinical treatments for retinal neovascularization, mainly intravitreal injection of anti-VEGF, retinal laser photocoagulation and vitrectomy. Commonly used treatments in clinical practice cannot cure the disease. Finding the mechanism pathway of retinal neovascularization, finding new targets, and treating retinal neovascularization through gene therapy are currently hot topics for fundamental treatment. Summary of the invention

[0006] Based on this, the purpose of the present invention is to provide an application of shRNA and a recombinant viral vector that inhibit BCL-9 in treating retinal angiogenesis-related diseases, wherein the shRNA and the recombinant viral vector can effectively inhibit retinal angiogenesis.

[0007] In a first aspect, the present invention provides a shRNA for inhibiting the expression of BCL-9 gene, wherein the nucleotide sequence of the shRNA is shown in SEQ ID NO.2 or SEQ ID NO.3.

[0008] The second aspect of the present invention provides a recombinant viral vector for inhibiting the expression of BCL-9 gene, wherein the recombinant viral vector contains the shRNA as described above.

[0009] In some embodiments, the backbone of the recombinant viral vector is a lentivirus, an adeno-associated virus or an adenovirus.

[0010] The third aspect of the present invention provides the use of the shRNA or recombinant viral vector described above in the preparation of a drug for treating retinal angiogenesis-related diseases.

[0011] In some embodiments, the use comprises reducing the expression of the ESM1 gene after inhibiting the expression of the BCL-9 gene.

[0012] In some embodiments, the retinal angiogenesis-related diseases include diabetic retinopathy, neovascular age-related retinopathy, and retinopathy of prematurity.

[0013] A fourth aspect of the present invention provides a drug for treating retinal angiogenesis-related diseases, wherein the active ingredient of the drug includes the shRNA and / or recombinant viral vector as described above.

[0014] In some embodiments, the drug is in the form of an injection.

[0015] In some embodiments, the drug is in the form of an ocular injection.

[0016] The fifth aspect of the present invention provides a method for inhibiting retinal angiogenesis for non-therapeutic purposes, comprising the following steps: introducing the shRNA and / or recombinant viral vector described above into a test sample, so that the expression of the BCL-9 gene in the test sample is inhibited.

[0017] Due to the particularity of the retina, there are currently limited effective methods for treating retinal neovascularization in clinical practice, mainly intravitreal injection of anti-VEGF protein, retinal laser photocoagulation, and vitrectomy. However, these commonly used treatment methods in clinical practice cannot provide a radical cure.

[0018] The present invention has discovered the target BCL-9 for gene therapy of retinal neovascular diseases through research, and screened and obtained shRNA that can efficiently target and inhibit the expression of BCL-9 gene, and constructed a recombinant viral vector containing the shRNA. According to the particularity of ocular medication, the recombinant viral vector can be prepared into an injection formulation and directly injected into the eye. The shRNA is introduced into human retinal vascular endothelial cells through the recombinant viral vector and specifically inhibits the expression of the BCL-9 gene, which can effectively reduce the activity, proliferation ability and tube-forming ability of human retinal vascular endothelial cells, thereby inhibiting retinal angiogenesis and treating retinal angiogenesis-related diseases, including diabetic retinopathy, neovascular age-related retinopathy and retinopathy of prematurity. The present invention provides a new target for the treatment of retinal angiogenesis-related diseases and develops a new idea.

[0019] Further studies have shown that, unlike the anti-angiogenesis strategy based on anti-VEGF protein, the present invention uses the BCL-9 gene as a therapeutic target, and directly introduces shBCL9#3 into human retinal vascular endothelial cells through injection to target and inhibit BCL-9 gene expression, which can effectively reduce the expression of endothelial cell-specific molecules (ESM1), while having no significant effect on the expression of vascular endothelial growth factor (VEGFA). This indicates that the shRNA specifically targeting the BCL-9 gene of the present invention is directly introduced into human retinal vascular endothelial cells through injection to effectively inhibit retinal angiogenesis by reducing the expression of ESM1. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Figure 2 is a structural diagram of each recombinant lentiviral vector.

[0021] Figure 2 This is a graph showing the results of knocking down the expression of BCL9 in HREC using a recombinant lentiviral vector.

[0022] Figure 3 The results of the detection of the number of tube connection points and the amount of tube formation in HREC cells with BCL9 knockdown.

[0023] Figure 4The results of VEGFA expression detection in HREC cells with BCL9 knockdown.

[0024] Figure 5 The results of the test show that knocking down BCL9 affects the activity of HREC cells.

[0025] Figure 6 The results of the test show that knocking down BCL9 affects the proliferation ability of HREC cells.

[0026] Figure 7 These are the results of detecting the expression of BCL9 around new blood vessels caused by hypoxia in the mouse retina.

[0027] Figure 8 This is the result of RNA-seq sequencing. DETAILED DESCRIPTION

[0028] The experimental methods in the following examples of the present invention, for which specific conditions are not specified, are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0029] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0030] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0031] Example 1

[0032] In this example, after knocking down BCL9 in human retinal microvascular endothelial cells (HREC) using a recombinant lentiviral vector, in vitro studies such as tube formation experiments, monoclonal crystal violet staining experiments, CCK-8 experiments, and qPCR were performed, and in vivo experiments were performed after constructing OIR model mice to illustrate the role of recombinant lentiviral vectors in regulating the tube formation of human retinal microvascular endothelial cells after knocking down BCL9. At the same time, molecular biological experiments were used to clarify the mechanism of action of BCL9 in affecting retinal neovascularization.

[0033] 1. Knockdown of BCL9 expression in HRECs using recombinant lentiviral vector

[0034] Recombinant lentiviral vector was used to specifically knock down the expression of BCL9 in HRECs.

[0035] Yunzhou Biotechnology (Guangzhou) Co., Ltd. was commissioned to connect the shRNA and NC sequence of the BCL9 gene with the lentiviral backbone to obtain a recombinant lentiviral vector. In this embodiment, a variety of shRNAs for the BCL9 gene were designed, and two shRNAs that can effectively inhibit the expression of the BCL9 gene in HREC were screened. Taking shRNA1 to 3 as an example, the nucleotide sequence of shRNA1 for the BCL9 gene is shown in SEQ ID NO.1, the nucleotide sequence of shRNA2 is shown in SEQ ID NO.2, the nucleotide sequence of shRNA3 is shown in SEQ ID NO.3, and the NC sequence is shown in SEQ ID NO.4.

[0036]

[0037] The recombinant lentiviral vectors containing the above shRNA1-3 and NC sequences were named shBCL9#1, shBCL9#2, shBCL9#3, and si-NC, respectively. The structures and numbers of the recombinant lentiviral vectors are as follows: Figure 1 As shown, the above-mentioned recombinant lentiviral vector was used for experiments.

[0038] 1.1 Experimental procedures

[0039] A. On the first day, HREC cells were cultured at 1.5×10 5 The cells were seeded in 6-well plates and cultured for 24 hours until about 50% of them were transfected with recombinant lentiviral vectors. The experimental groups were set as: blank control group (control), blank vector group (si-NC), BCL9 knockdown group (three knockdown sequences: shBCL9#1, shBCL9#2, shBCL9#3), to verify the knockdown effects of the three different knockdown sequences.

[0040] B. On the second day, mix the complete culture medium and the transfection reagent (Hitrans GA): culture medium volume = number of wells × 960ul transfection reagent volume + number of wells × 40ul. Mix the culture medium and transfection reagent evenly and add 1ml of the mixture to each well.

[0041] C. Calculate the volume of virus added: the volume of virus added to each well = number of cells * MOI / titer. The MOI value is 10. That is, the volume of virus added to each well = (number of cells × 10) / (virus titer). Add the corresponding volume of virus to each well.

[0042] D. After 24 hours of incubation at 37°C, replace with fresh culture medium and add 2 ml to each well. 48 hours after transfection, cells can be extracted for further experiments.

[0043] E. Extract the transfected cells, extract RNA (QIAGEN, 74104), reverse transcribe (Takara, RR036A), and then use qPCR (Takara, RR420A) to verify the knockdown effect of BCL9 in the cells. The operation is strictly carried out according to the instructions. The detection primers for the BCL9 gene are: Upstream primer: Downstream primer:

[0044] 1.2 Experimental Results

[0045] Results: Figure 1 As shown, compared with the control group, green fluorescence appeared in a large number of HRECs in the si-NC group and the shBCL9#3 group, indicating that the recombinant lentiviral vector was successfully transfected.

[0046] qPCR test results ( Figure 2 ) showed that both shhBCL9#2 and shBCL9#3 knockdown groups could effectively knock down BCL9 expression in HRECs, while shBCL9#1 had no significant knockdown effect. Among them, the knockdown efficiency of shBCL9#3 knockdown group was greater than 50%, which was more effective in knocking down BCL9 expression in HRECs than other shRNAs.

[0047] 2. Investigate the effect of knocking down BCL9 gene expression on the tube-forming ability of HREC.

[0048] 2.1 Experimental procedures

[0049] A, HRECs were transfected with recombinant lentiviral vector for 48 hours. Groups: blank control group (control), blank vector group (si-NC), BCL9 knockdown group (shBCL9#3).

[0050] B. Coming MatrigelT M Incubate the basement membrane matrix (Catalog No. 354230) on ice for at least 10 minutes until the matrix gel becomes liquid.

[0051] C. Tube formation experiment was performed on u-slide angiogenesis slide (ibidi, 81506), 10 μL Matrigel was added to each well, and then incubated at 37°C for 30 minutes.

[0052] D. Digest the cells 48 hours after transfection and adjust the cell density to 2×105 / ml, and prepare cell suspension.

[0053] E. Add 50ul / well of cell suspension (10,000 cells / well) on top of the Matrige. After incubation at 37°C for 1h, 6h, and 24h, take a picture of each well under a microscope.

[0054] F. The number of endothelial lumens in three replicate wells was counted using Angiogenesis Analyzer in ImageJ version 1.48 software and statistical analysis was performed. Data are expressed as mean ± SD. One-way ANOVA was used for statistical analysis (*P < 0.05).

[0055] 2.2 Experimental Results

[0056] Results: Figure 3 As shown, 6 hours after plating, the number of tube-forming connection points and the amount of tube-forming in HREC cells after knocking down BCL9 in the shBCL9#3 group were significantly reduced compared with those in the si-NC group, that is, the tube-forming ability of HREC cells after knocking down BCL9 was significantly reduced.

[0057] 3. Effect of recombinant lentiviral vector transfection and knockdown of BCL9 in HREC on VEGFA

[0058] 3.1 Experimental procedures

[0059] (1) HREC were transfected with recombinant lentiviral vector for 48 hours. Groups: blank control group (control), blank vector group (si-NC), BCL9 knockdown group (shBCL9#3).

[0060] (2) MOI value of lentivirus transfection: 10. Take fresh complete medium, add diluted lentivirus and mix well. Add 1 ml of complete medium containing lentivirus to each well. Add 1 ml of complete medium without lentivirus to the blank control group. Place the 6-well plate in an incubator at 37°C, 5% CO2 and culture for 48 hours.

[0061] (3) HREC RNA was extracted 48 hours after transfection, and the expression of BCL9 and VEGFA genes after lentiviral transfection was detected by qPCR after reverse transcription. The detection kit was the same as above. The detection primers for VEGFA gene were: Upstream primer: Downstream primer:

[0062] 3.2 Experimental Results

[0063] Results: Figure 4As shown in Figure A, compared with the si-NC group, BCL9 in HRECs of the shBCL9#3 knockdown group was effectively knocked down, but there was no statistical difference in the expression of VEGFA ( Figure 4 (B). Therefore, the effect of shBCL9#3 knockdown on BCL9 on retinal angiogenesis was not significantly correlated with VEGFA.

[0064] 4. CCK-8 test to examine the effect of knocking down BCL9 on the cell activity of HREC

[0065] 4.1 Experimental procedures

[0066] A. HRECs were transfected with recombinant lentiviral vector for 48 hours. Groups: blank control group (control), blank vector group (si-NC), BCL9 knockdown group (shBCL9#3). Lentivirus transfection MOI value: 10.

[0067] B. Digest the cells and make a cell suspension. Use a spray gun to seed the cells into a 96-well plate, with 100ul of cell suspension per well and a cell density of 1.5×10 5 / ml. Add 100ul PBS to each well around the cell well to prevent the culture medium from evaporating. Place the 96-well plate in an incubator at 37°C, 5% CO2, and culture for 48h and 5 days respectively, replacing fresh complete culture medium every two days.

[0068] C. After culturing for 48 h and 5 days respectively, take out the 96-well plate, aspirate the original culture medium, replace it with 90 ul of fresh culture medium, and add 90 ul of fresh complete culture medium to the wells without cells as CCK 8 blank control wells. Then add 10 ul of CCK8 reagent to each well, protect from light, and place in an incubator at 37°C, 5% CO2 for incubation for 2 h.

[0069] D. Take out the culture plate and protect it from light. Test the absorbance at 450 nm on an ELISA reader.

[0070] E. Export data analysis and calculate cell viability. The calculation formula is as follows:

[0071] Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%

[0072] As: absorbance of experimental wells (containing knockdown BCL9 cells, culture medium, and CCK-8 solution);

[0073] Ac: absorbance of control well (containing NC group cells, culture medium, and CCK-8 solution);

[0074] Ab: absorbance of blank wells (containing culture medium and CCK-8 solution, but without cells and drugs).

[0075] 4.2 Experimental Results

[0076] Results: Figure 5 As shown, after knocking down BCL9, the cell activity of HREC was 0.6235 of that of the blank vector group after 48 hours (P < 0.0001), and the cell activity after 5 days was 0.1977 of that of the blank vector group (P < 0.0001), that is, after knocking down BCL9, the cell activity of HREC was significantly reduced.

[0077] 5. Crystal violet staining of monoclonal cells to observe the effect of knockdown of BCL9 on the proliferation ability of HREC

[0078] 5.1 Experimental procedures

[0079] A, HRECs were transfected with recombinant lentiviral vector for 48 hours. Groups: blank control group (control), blank vector group (si-NC), BCL9 knockdown group (shBCL9#3).

[0080] B. Prepare cell suspension, perform cell technology, and seed the cells into 6-well plates at 1000 cells per well according to the group. Add complete culture medium to 3 ml per well.

[0081] C. Place the cells in a cell culture incubator at 37°C and 5% CO2 for 5 days.

[0082] D. After 5 days of normal culture, obvious cell clones can be seen. Take out the 6-well plate, discard the original culture medium, and carefully wash the cells 3 times with PBS. Aspirate off the cBS.

[0083] E. Add 1 ml of methanol to each well, fix the cells for 15 minutes, and discard the methanol.

[0084] F. Add 1 ml of 0.1% crystal violet to each well and stain for 30 minutes. Remove the crystal violet by aspiration, wash the cells three times with PBS, and dry in the air.

[0085] G. Take photos under a microscope and count the cloned cells. The experiment was repeated 4 times. Statistical analysis was performed. All data are expressed as mean ± SD. One-way ANOVA was used for statistical analysis (*P < 0.05).

[0086] 5.2 Experimental Results

[0087] Results: Figure 6 As shown in the figure, after knocking down BCL9 in HREC cells, the number of cells in monoclonal clusters was significantly reduced compared with the blank control group and the blank vector group, and the number of surviving monoclonal cell clusters was significantly reduced compared with the control group. That is, after knocking down BCL9, the proliferation ability of HREC was significantly reduced (P < 0.001).

[0088] 6. Effect of BCL9 on neovascularization in hypoxia-induced retinal (OIR) mice

[0089] 6.1 Experimental procedures

[0090] (1) Preparation of OIR model mice: Newborn C57BL / 6J pups and their nursing mothers were placed in an oxygen chamber and subjected to alternating high and low oxygen cycles (50%±2% and 10%±2% O2, respectively) every day for 14 days. The mice were then returned to room air until they were 18 days old.

[0091] (2) Soak the mouse eyeball in 4% paraformaldehyde for 1 hour, replace with pre-cooled PBS, and place on ice. Use a 30G needle to puncture the cornea to reduce intraocular pressure, cut off a circle of cornea and remove the lens and vitreous body, leaving the ciliary body as much as possible when cutting the cornea, carefully peel off the choroid and retina, and then cut the retina into four radial petals and transfer them to a slide, while soaking the retina in frozen PBS.

[0092] (3) Incubate the tissue with 1% Triton-X (1:100 Triton-X:PBS) for 30 minutes at room temperature and wash the tissue three times with PBS.

[0093] (4) Block the retina with 5% blocking solution at room temperature for 1 hour at 4°C.

[0094] (5) 1% Isolectin B4 (4 μg / mL, Alexa Fluor 488, catalogue no. I21411; Life Technologies Australia, Mulgrave, VIC, Australia) and anti-BCL9 primary antibody (abcam, ab37305) were diluted in 5% blocking solution at a ratio of 1:500, overlaid on the retina, and incubated overnight at 4°C in the dark.

[0095] (6) The next day, the retina was washed three times with PBS and incubated with secondary antibody for 2 h at room temperature.

[0096] (7) Wash the retina three times with PBS, wipe the water on the slide with a diameter, seal the slide with anti-fluorescence quenching mounting medium containing DAPI and transparent nail polish, and take pictures under a fluorescence microscope.

[0097] 6.2 Experimental Results

[0098] Results: Figure 7As shown in the figure, hypoxia-induced neovascularization (red) occurred in the mouse retina, and we found that BCL9 (green) was highly expressed around the neovascularization. That is, the expression of BCL9 is positively correlated with the formation of neovascularization.

[0099] 7. RNA-sequencing (RNA-seq) technology detects that BCL9 can affect the ability of angiogenesis through ESM1

[0100] 7.1 Experimental procedures

[0101] (1) Cell culture and grouping: HRECs were transfected with recombinant lentiviral vectors for 48 hours. Grouping: blank control group (control), blank vector group (si-NC), BCL9 knockdown group (shBCL9#3).

[0102] (2) 72 hours after lentiviral transfection, cells were harvested and RNA was extracted.

[0103] (3)RNA-seq method

[0104] RNA-seq was commissioned to Genewise Biotechnology Co., Ltd. and included the following steps:

[0105] 1) 1ug of total RNA was used for the preparation of the following library.

[0106] 2) Isolation of poly(A) mRNA using Oligo(dT) beads.

[0107] 3) Use divalent cations and high temperature to fragment mRNA.

[0108] 4) Primer screening using random primers.

[0109] 5) Synthesize the first-strand cDNA and the second-strand cDNA, then treat the purified double-stranded cDNA, repair both ends and add dA-tailing in one reaction, followed by TA ligation and adding adapters to both ends.

[0110] 6) Size selection of the adapter-ligated DNA was performed using DNA cleaning beads. Each sample was then PCR amplified using primers P5 and P7, and the PCR products were verified.

[0111] 7) Libraries with different indices were multiplexed and loaded onto an Illumina HiSeq / Illumina Novaseq / MGI2000 instrument and sequenced using a 2x150 paired-end (PE) configuration according to the instrument's instructions.

[0112] 7.2 Experimental Results

[0113] Results: RNA-seq sequencing was used to analyze the differentially expressed genes in HRECs after shBCL9#3 knocked down BCL9 expression in HRECs. We analyzed the top 20 mRNAs with reduced expression. The results showed that the endothelial cell-specific molecule (ESM1) associated with angiogenesis was reduced ( Figure 8 , NC-1 to NC-4 in the figure represent four repeated NC samples, and shBCL9-1 to shBCL9-4 represent four repeated shBCL9#3 samples). Therefore, it can be seen that, unlike the anti-angiogenesis strategy based on anti-VEGF protein, the present invention uses the BCL-9 gene as a therapeutic target, and directly introduces shBCL9#3 into human retinal vascular endothelial cells through injection to target and inhibit BCL-9 gene expression, which can effectively reduce the expression of ESM1 and effectively inhibit the formation of retinal angiogenesis at the gene level.

[0114] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0115] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A shRNA for inhibiting BCL-9 gene expression, characterized in that: The nucleotide sequence of the shRNA is shown as SEQ ID NO.2, or as SEQ ID NO.

3.

2. A recombinant viral vector for inhibiting BCL-9 gene expression, characterized in that: The recombinant viral vector contains the shRNA according to claim 1.

3. The recombinant viral vector according to claim 2, characterized in that The backbone of the recombinant viral vector is lentivirus, adeno-associated virus or adenovirus.

4. Use of the shRNA according to claim 1 or the recombinant viral vector according to any one of claims 2 to 3 in the preparation of a drug for treating retinal angiogenesis-related diseases.

5. The use according to claim 4, characterized in that The application includes reducing the expression of ESM1 gene after inhibiting the expression of BCL-9 gene.

6. The use according to claim 4 or 5, characterized in that The retinal angiogenesis-related diseases include diabetic retinopathy, neovascular age-related retinopathy, and retinopathy of prematurity.

7. A drug for treating retinal angiogenesis-related diseases, characterized in that: The active ingredient of the drug includes the shRNA according to claim 1 and / or the recombinant viral vector according to any one of claims 2 to 3.

8. The drug according to claim 7, characterized in that The dosage form of the drug is injection.

9. The drug according to claim 8, characterized in that The dosage form of the drug is an ocular injection.

10. A method for inhibiting retinal angiogenesis for non-therapeutic purposes, characterized in that: The following steps are involved: The shRNA according to claim 1 or the recombinant viral vector according to any one of claims 2 to 3 is introduced into a test sample to inhibit the expression of the BCL-9 gene in the test sample.

Images