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Definitions

• the present invention relates to the fields of immunology and gene delivery. More specifically, the present application relates to compositions, systems, and methods for producing proteins of interest, such as antibodies.
• Age-related macular degeneration is a group of macular diseases induced by various factors and related to age. Its common feature is the occurrence of lesions in the macular retina, retinal pigment epithelium and choroid, which leads to visual impairment in patients. Functional impairment and progressive decrease in central vision. The prevalence of AMD in our country and even around the world continues to rise and increases with age. It is one of the main causes of irreversible visual impairment in people over 50 years old. As the population ages, the number of AMD patients is expected to reach 288 million by 2040. There is no unified standard for the clinical classification of AMD. At present, both domestic and foreign countries tend to stage first and then classify.
• Neovascular age-related macular degeneration also known as wet age-related macular degeneration (wAMD)
• CNV choroidal neovascularization
• DME diabetic macular edema
• VEGF Vascular endothelial growth factor
• drugs targeting VEGF have made great clinical progress and breakthroughs, they can significantly reduce the degree of vascular leakage and edema, improve vision, and no serious complications have been found.
• these protein drugs injected into the body are rapidly eliminated through metabolism, requiring multiple intraocular injections to maintain therapeutic effects. Long-term treatment increases the patient's economic burden. Repeated injections will increase the patient's pain and the possibility of adverse reactions. There will be a certain degree of vision loss when switching from regular dosing to low-frequency dosing. Some patients are prone to relapse after treatment. .
• In response to the limitations of existing therapies for wAMD and DME there is a need in the field for more economical, longer-lasting, and more effective treatment strategies.
• the present invention aims to provide an anti-VEGF-A and anti-ANG-2 gene therapy.
• the invention provides a bispecific antibody, which includes an anti-VEGF-A and ANG-2 binding domain, which is preferably a single-chain antibody tandem molecule (tandem scFv, ta-scFv).
• the chain variable region (VH) and light chain variable region (VL) are arranged in the following order from N-terminus to C-terminus:
• the VH anti-VEGF-A includes CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2; CDR3 shown in SEQ ID NO:3;
• the VL anti-VEGF-A includes CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5; CDR3 shown in SEQ ID NO:6 or SEQ ID NO:47;
• the VH anti-ANG-2 includes CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8; CDR3 shown in SEQ ID NO:9;
• the VL anti-ANG-2 includes CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11; CDR3 shown in SEQ ID NO:12.
• the VH anti-VEGF-A includes the sequence shown in SEQ ID NO:13 or has a sequence identity of 70%, 80%, 90%, 95%, or 99% with SEQ ID NO:13 sequence;
• the VL anti-VEGF-A includes the sequence shown in SEQ ID NO:14 or has a sequence identity of 70%, 80%, 90%, 95%, or 99% with SEQ ID NO:14 or SEQ ID NO:48 sequence;
• the VH anti-ANG-2 includes the sequence shown in SEQ ID NO: 15 or a sequence with a sequence identity of 70%, 80%, 90%, 95%, or 99% to SEQ ID NO: 15;
• the VL anti-ANG-2 includes the sequence shown in SEQ ID NO: 16 or a sequence with a sequence identity of 70%, 80%, 90%, 95%, or 99% to SEQ ID NO: 16.
• the antibody includes the sequence shown in SEQ ID NO: 17, 18, 19, 49, 51 or 53 or has a sequence identity of SEQ ID NO: 17, 18, 19, 49, 51 or 53. 70%, 80%, 90%, 95%, or 99% of the sequence.
• the N-terminus of the bispecific antibody construct includes a signal peptide sequence or a tag sequence, preferably the signal peptide sequence is a CD5-sp signal peptide, which includes as shown in SEQ ID NO: 23 sequence.
• the antibody includes the sequence shown in SEQ ID NO: 20, 21, 22, 50, 52 or 54 or has a sequence identity of SEQ ID NO: 20, 21, 22, 50, 52 or 54. 70%, 80%, 90%, 95%, or 99% of the sequence.
• the heavy chain variable region (VH) and the light chain variable region (VL) are operably linked by (G 4 S) n , where n is an integer greater than 1, preferably 1 to Any integer between 4, such as G 4 S, (G 4 S) 2 , (G 4 S) 3 or (G 4 S) 4 , for example,
• VL anti-ANG-2- (G 4 S) m -VH anti-VEGF-A- (G 4 S) m+X -VL anti-VEGF-A- (G 4 S) m -VH anti-ANG-2 , where m ⁇ 1 , _ _ _ _ _ _ _ _
• the present invention provides a nucleic acid sequence encoding the aforementioned bispecific antibody.
• the present invention also provides a vector including a nucleic acid sequence encoding the aforementioned bispecific antibody.
• the vector is preferably an AAV virus.
• the AAV viral vector further includes: 5'ITR and 3'ITR, a promoter and polyA sequence.
• the invention provides an AAV virus particle, including any of the above AAV viral vectors and capsid proteins, preferably the serotypes of the capsid proteins are AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9.
• the present invention provides a pharmaceutical composition, including at least one of the bispecific antibody as described above, a nucleic acid sequence, a vector such as an AAV viral vector and an AAV virus particle as described above, and a pharmaceutically acceptable carrier.
• the present invention provides bispecific antibodies, nucleic acid sequences, vectors such as AAV viral vectors, AAV viral particles as described above, or pharmaceutical compositions as described above for use in the treatment or prevention of cancer, intraocular Use in medicines for neovascular syndrome, rheumatoid arthritis, psoriasis, proliferative retinopathy, age-related macular degeneration, or diabetic macular edema.
• vectors such as AAV viral vectors, AAV viral particles as described above, or pharmaceutical compositions as described above for use in the treatment or prevention of cancer, intraocular Use in medicines for neovascular syndrome, rheumatoid arthritis, psoriasis, proliferative retinopathy, age-related macular degeneration, or diabetic macular edema.
• the age-related macular degeneration is wet age-related macular degeneration.
• the drug is administered by intravitreal or subretinal injection.
• Figure 1 shows the structural schematic diagram of XMVA01, XMVA04 and XMVA09.
• Figure 2 shows the vector information of pAAV9-XMVA09 and ssAAV-XMVA09.
• Figure 3 shows the expression of VEGF165 neutralizing protein after cells were transfected with pAAV9-XMVA01, pAAV9-XMVA04 and pAAV9-XMVA09 vectors.
• Figure 4 shows the effect of expressing XMVA01, XMVA04 and XMVA09 in cells on the proliferation of human retinal microvascular endothelial cells (HRMEC) under ECGS stimulation.
• HRMEC retinal microvascular endothelial cells
• Figures 5 and 6 show the effects of expressing XMVA01, XMVA04 and XMVA09 in cells on the lumen formation of HRMECs.
• Figure 7 shows a schematic structural diagram of XMVA10.
• Figure 8 shows a schematic structural diagram of XMVA11.
• Figure 9 shows a schematic structural diagram of XMVA13.
• Figure 10 shows a schematic structural diagram of XMVA14.
• Figure 11 shows a schematic structural diagram of XMVA15.
• Figure 12 shows fluorescein angiography (FFA) images of laser-induced wAMD model mice after injection of AAV-XMVA09.
• Figure 13 shows the level 3 spot percentage and average spot leakage score in laser-induced wAMD model mice after injection of AAV-XMVA09.
• Figure 14 shows the changes in the number of level IV spots and fluorescence leakage area in the laser-induced wAMD model rhesus monkey control group.
• Figure 15 shows the changes in the number of grade IV spots and fluorescence leakage area in rhesus monkeys injected with AAV-XMVA09 in the laser-induced wAMD model.
• Figure 16 shows the changes in retinal thickness of level IV spots in the laser-induced wAMD model rhesus monkey control group.
• Figure 17 shows the changes in retinal thickness of grade IV spots in laser-induced wAMD model rhesus monkeys injected with AAV-XMVA09.
• Figure 18 shows the permeability changes of HRMECs induced by high glucose.
• Figure 19 shows the regulatory effect of XMVA09 on the permeability of HRMECs based on the VE-cadherin index.
• Figure 20 shows the judgment of XMVA09’s effect on HRMECs based on biotin-avidin system indicators. permeability control effect.
• Figure 21 shows the monitoring analysis of fasting blood glucose and body weight of diabetic model mice.
• Figure 22 shows images of retinal blood vessel leakage in diabetic model mice.
• Figure 23 shows changes in retinal blood vessel leakage area in diabetic model mice after injection of AAV-XMVA09.
• the antibodies in the present invention are multispecific and can be humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutant, and grafted antibodies; the antibody form in the present invention is An scFv is spliced into an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
• VL antibody light chain variable region
• VH antibody heavy chain variable region
• the antibody can bind to two different antigens, such as VEGF-A and ANG-2.
• VL Antibody light chain variable region
• VH antibody heavy chain variable region
• CDRs complementarity-determining regions
• FWR interspersed regions
• the CDRs of the antibodies and antigen-binding fragments disclosed herein are defined or identified by Kabat numbering.
• each VH and VL generally includes 3 CDRs and 4 FWRs arranged from amino terminus to carboxyl terminus in the following order: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4.
• the CDRs of the antibodies and antigen-binding fragments disclosed herein are defined or identified by Kabat numbering.
• vector refers to a vector that includes a recombinant polynucleotide that includes expression control sequences operably linked to the nucleotide sequence to be expressed.
• VH amino acid sequence and VL amino acid sequence of anti-VEGF are added to the N-terminus, using (G 4 S) 3 and G respectively.
• (G 4 S) 3 peptide linkers are connected to form a structure of CD5-sp-VH anti-VEGF-A - (G 4 S) 3 -VL anti-VEGF-A -G 4 S-VH anti-ANG-2 -(G 4 S) 3- VL anti-ANG-2 open reading frame, nucleotide sequence designed according to human codon preference , and introduced a BamHI restriction site at the 5' end and an EcoR V restriction site at the 3' end.
• Example 2 Determination of expression of XMVA01, XMVA04, and XMVA09 in cells
• pAAV9-XMVA01, pAAV9-XMVA04, and pAAV9-XMVA09 plasmids were transfected into HEK293T cells respectively, the supernatant was collected, and the protein expression in the supernatant was detected by ELISA.
• the specific operation is as follows: resuspend the 293T cells in complete culture medium of DMEM containing 10% fetal bovine serum, inoculate them into a culture dish, and culture them in a 37°C, 5% CO2 incubator until the confluence of the cells reaches 70%-90%.
• Lipofectamine 2000 (Invitrogen) transfection reagent the plasmid was transfected according to its instructions. After continuing to culture for 72 hours, the supernatant was collected and stored at -80°C for later use.
• the specific detection method of ELISA is as follows: Coat the enzyme plate (Thermo) with 0.1 ⁇ g/mL VEGF165 (proteintech) protein, 100 ⁇ L per well, incubate at 4°C overnight, and wash the enzyme plate three times with PBS containing 0.05% Tween (Sangon) , 3 minutes each time. Block with 200 ⁇ L/well of PBS containing 2% BSA (Maike Biotech) for 1 hour at room temperature, and wash three times again.
• Example 3 Effects of expressing XMVA01, XMVA04, and XMVA09 in cells on the proliferation of human retinal microvascular endothelial cells (HRMEC)
• HRMECs were conditioned with complete medium (Science Cell) in ECM containing 1% ECGS The cell density is 4 ⁇ 10 4 /mL, and 100 ⁇ L/well is seeded in a flat-bottom 96-well plate; the cell supernatant collected after transfecting HEK293T cells with pAAV9-XMVA01, pAAV9-XMVA04, and pAAV9-XMVA09 plasmids is 100 ⁇ L/well. Add into the well, use the cell supernatant without transfected plasmid as the control group, and culture it in a 37°C, 5% CO2 incubator for 72 hours; follow the Cell Counting Kit-8 (CCK-8, Biosharp) instructions.
• Example 4 Effects of expressing XMVA01, XMVA04, and XMVA09 in cells on the lumen formation of HRMECs
• VH amino acid sequence and VL amino acid sequence of the anti-VEGF, the VH amino acid sequence and the VL amino acid sequence of the anti-ANG-2, and the secretion signal peptide CD5-sp nucleotide sequence are added to the N-terminus, using G 4 S, (G 4 S) 3 and G 4 S peptide linkers are connected to form the structure CD5-sp-VL anti - ANG-2 -G 4 S-VH anti-VEGF-A -(G 4 S) 3 -VL anti-VEGF-A -G 4
• the S-VH anti-ANG-2 open reading frame was designed with a nucleotide sequence based on human codon preference, and a BamHI cleavage site was introduced at the 5' end and an EcoR V cleavage site was introduced at the 3' end, named XMVA10 (see Figure 7). Among them, anti-VEGF The VH amino acid sequence, VL amino acid sequence, and anti-ANG-2 VH amino acid sequence
• VH amino acid sequence and VL amino acid sequence of anti-VEGF are added to the N-terminus, using G 4 S and (G 4 S respectively.
• G 4 S peptide linkers are connected to form the structure CD5-sp-VL anti-VEGF-A -G 4 S-VH anti-ANG-2 -(G 4 S) 3 -VL anti-ANG-2 -G 4 S -VH anti-VEGF-A open reading frame, the nucleotide sequence is designed according to human codon preference, and a BamHI cleavage site is introduced at the 5' end and an EcoR V cleavage site is introduced at the 3' end, named XMVA11 (See Figure 8).
• the VH amino acid sequence and VL amino acid sequence of anti-VEGF, and the VH amino acid sequence and VL amino acid sequence of anti-ANG-2 are the same as XMVA09.
• VH amino acid sequence and VL amino acid sequence of anti-VEGF, the VH amino acid sequence and VL amino acid sequence of anti-ANG-2, and the secretion signal peptide CD5-sp nucleotide sequence are added to the N terminus, using (G 4 S) 3 and G 4 S and (G 4 S) 3 peptide linkers are connected to form the structure CD5-sp-VH anti-VEGF-A -(G 4 S) 3 -VL anti-VEGF-A -G 4 S-VH anti-ANG-2 -(G 4 S) 3- VL anti-ANG-2 open reading frame, the nucleotide sequence is designed according to human codon preference, and a BamHI cleavage site is introduced at the 5' end and an EcoR V enzyme is introduced at the 3' end The cleavage site was named XMVA13 (see Figure 9).
• VH amino acid sequence and VL amino acid sequence of the anti-VEGF, the VH amino acid sequence and the VL amino acid sequence of the anti-ANG-2, and the secretion signal peptide CD5-sp nucleotide sequence are added to the N-terminus, using G 4 S, (G 4 S) 3 and G 4 S peptide linkers are connected to form the structure CD5-sp-VL anti - ANG-2 -G 4 S-VH anti-VEGF-A -(G 4 S) 3 -VL anti-VEGF-A -G 4
• the S-VH anti-ANG-2 open reading frame was designed with a nucleotide sequence based on human codon preference, and a BamHI cleavage site was introduced at the 5' end and an EcoR V cleavage site was introduced at the 3' end, named XMVA14 (see Figure 10).
• VH amino acid sequence and VL amino acid sequence of anti-VEGF, the VH amino acid sequence and VL amino acid sequence of anti-ANG-2, plus the secretion signal peptide CD5-sp nucleotide sequence at the N-terminus They were connected with G 4 S, (G 4 S) 3 and G 4 S peptide linkers respectively to form the structure CD5-sp-VL anti-VEGF-A -G 4 S-VH anti-ANG-2 -(G 4 S) 3 -VL anti-ANG-2 -G 4 S-VH anti-VEGF-A open reading frame, the nucleotide sequence is designed according to human codon preference, and a BamHI restriction site is introduced at the 5' end and a BamHI restriction site is introduced at the 3' end
• the EcoR V restriction site is named XMVA15 (see Figure 11).
• the VH amino acid sequence and VL amino acid sequence of anti-VEGF, and the VH amino acid sequence and VL amino acid sequence of anti-ANG-2 are
• the XMVA10, XMVA11, XMVA13, XMVA14 and XMVA15 vectors were constructed through conventional molecular biology operations such as ligation, transformation and clone screening and identification, and high-quality plasmid DNA was obtained using an endotoxin-free plasmid extraction kit (MN) for later use.
• MN endotoxin-free plasmid extraction kit
• Example 2 the plasmids were transfected into HEK293T cells, the supernatant was collected, and the protein expression in the supernatant was detected by ELISA. According to the method described in Example 3, the effect of expressing the above vector in cells on the proliferation of HRMECs was evaluated. According to the method described in Example 4, the effect of expressing the above plasmid in cells on the lumen formation of HRMECs was evaluated. Recombinant AAV viruses were prepared and characterized according to the method described in Example 5.
• Example 6 Preparation and identification of recombinant AAV viruses
• An endotoxin-free plasmid extraction kit MN
• MN endotoxin-free plasmid extraction kit
• AAV virus including a helper plasmid (phhelper), AAV Cap and Rep protein expression plasmids, and a plasmid expressing the target gene ( ssAAV-XMVA09) and PEI transfection promoter to form a transfection complex according to the mass ratio of 2:1:1, transfect HEK293T cells, and perform AAV-XMVA09 virus packaging.
• the supernatant was collected twice to obtain AAV virus particles containing the target gene.
• Example 7 Inhibitory effect of XMVA09 on laser-induced CNV in wAMD model mice
• High-energy laser photocoagulation of the retina is a commonly used CNV animal model at home and abroad, and it is also the standard animal model in most current treatment evaluation experiments. It selectively destroys the photoreceptor outer segment disc membrane, Bruch's membrane, retinal pigment epithelium and part of the anterior choriocapillaris network, followed by a damage repair reaction, including the invasion and growth of fibroblasts, retinal pigment epithelial cells and vascular endothelial cells, and finally in the light New blood vessels form in the coagulated area.
• mice CNV model 1-2 drops of compound tropicamide eye drops were dropped into each eye of the mouse to dilate the pupils, and 5% chloral hydrate was injected intramuscularly for anesthesia. After anesthesia, carbomer eye drops were instilled in both eyes, a fundus laser lens was placed, and photocoagulation was performed around the optic papilla at a distance of about 1.5-2 PD from the optic disc to avoid blood vessels.
• the laser parameters are: wavelength 532nm, power 80mW, spot diameter 50 ⁇ m, exposure time 100ms. After photocoagulation, the animal's eyes are coated with erythromycin eye ointment.
• mice were subjected to fluorescein fundus angiography (FFA): intraperitoneal injection of fluorescein sodium injection (15 mg/mL, 10 mL/kg), early (within 1.5 minutes), late (3 Minutes later) collect several clear pictures with both eyes, rate the degree of fluorescence leakage of the effective spot, and calculate the percentage of level 3 leakage spots and the average score of spot leakage.
• FFA fluorescein fundus angiography
• An effective spot refers to a spot that does not have severe retinal hemorrhage nearby and can be completely displayed in FFA.
• Spot fluorescence leakage rating standards are: Level 0 (no fluorescence leakage), Level 1 (mild fluorescence leakage, leakage area is 1 to 50% of the laser spot size), Level 2 (moderate fluorescence leakage, leakage The leakage area is 50-100% of the laser spot size), level 3 (severe fluorescence leakage, the leakage area is larger than the laser spot size).
• the percentage of light spots at each level (%) the total number of light spots at the corresponding level ⁇ the total number of 4 types of light spots ⁇ 100%.
• the average score of light spot leakage [(number of level 0 light spots ⁇ 0) + (number of level 1 light spots ⁇ 1) + (number of level 2 light spots ⁇ 2) + (number of level 3 light spots ⁇ 3)] ⁇ the total number of 4 types of light spots.
• Example 8 Inhibitory effect of XMVA09 on CNV in laser-induced wAMD model rhesus monkeys
• Group XMVA09 AAV-XMVA09 (50 ⁇ L/eye) was injected into the vitreous cavity of both eyes 21 days before laser modeling, and CNV was induced by fundus laser in both eyes on day 0.
• Control group CNV was induced by fundus laser in both eyes on day 0, and PBS (50 ⁇ L/eye) was injected into both eyes on day 21.
• the animal's head is fixed in front of the ophthalmic laser photocoagulation instrument.
• laser photocoagulation is performed at a distance from the optic disc around the center of the macula, with 9 points in each eye.
• the laser parameters are: wavelength 532nm, power 650mW-700mW, spot diameter 50 ⁇ m, exposure time 0.1 seconds.
• FP and FFA measure the fluorescein leakage area.
• the specific inspection methods are as follows: (1) The animals are anesthetized by intramuscular injection of ketamine hydrochloride injection (20 mg/kg) and dexmedetomidine hydrochloride injection (0.03 mg/kg).
• grading standards for fluorescein leakage from laser spots are: Level I (spot without high fluorescence), Level II (spot with high fluorescence but no leakage), Level III (spot with high fluorescence and slight fluorescein leakage, but no leakage). beyond the edge of the spot), Level IV (high fluorescence in the spot, significant fluorescein leakage, and leakage beyond the edge of the spot).
• OCT measures changes in retinal thickness.
• the specific examination methods are as follows: (1) After the animal completes the FP and FFA examination, place it in front of the OCT instrument and adjust the animal's eyes to look straight at the scanning lens. (2) Use the follow-up mode to perform multi-layer linear scanning with the macula as the center, and the scanning area covers all laser points. (3) Select the layer where the retinal thickness changes most obviously and the retinal boundary is clear after modeling, and use the measurement software that comes with the instrument to measure the maximum retinal thickness in this area as an OCT efficacy evaluation index.
• Immunofluorescence staining steps Discard the culture supernatant and leave 200 ⁇ L of culture medium. Add 200 ⁇ L of 4% PFA to start gradient fixation and fix at room temperature for 20 minutes. After discarding the supernatant, directly add 200 ⁇ L of PFA and continue fixation at room temperature for 10 minutes.
• Example 10 Determining the regulatory effect of XMVA09 protein on HRMECs permeability based on VE-cadherin index
• Example 11 Determining the regulatory effect of XMVA09 protein on the permeability of HRMECs based on biotin-avidin system indicators
• the supernatant of cells transfected with ssAAV-XMVA09 plasmid expressing HEK293T was incubated with high sugar (30mM glucose), and HRMECs were treated for 17 hours.
• the control group was a non-modeling group without high sugar treatment; stained with FITC-avidin , and then fixed with 4% PFA, started staining with AF594-phalloidin (Invitrogen A12381), then added an anti-fluorescence quencher containing DAPI, and took photos and analysis under a fluorescence microscope.
• the results are shown in Figure 20.
• the protein expressed by ssAAV-XMVA09 has a significant inhibitory effect on the increase in permeability of HRMECs treated with high glucose.
• Drug induction is currently one of the methods for establishing diabetic animal models, which can better simulate the clinical pathological manifestations and clinical characteristics of diabetes.
• Streptozotocin (STZ) is the most commonly used drug to induce diabetes. It mainly causes hyperglycemia by destroying pancreatic beta cells, which in turn causes the loss of capillary pericytes, thinning of the vascular layer, and destruction of the blood-retina barrier, further leading to vascular leakage. Leak formation is a good model for studying DME.
• mice 80 male C57BL/6J mice of SPF grade 6 to 8 weeks old (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.), weighing 20-25g, were randomly divided into blank group, DME modeling control group, and DME Build a model set. Before modeling, fasting blood glucose and body weight of mice in the three groups were measured: fasted for 6 hours (since After drinking water), blood was collected from the tip of the tail for blood glucose measurement and weighed. This was the 0th day of modeling. Modeling was started on the first day. The mice were fasted (drinking water freely) for 6 hours. The blank group was not treated.
• the DME modeling control group was injected with a single intraperitoneal injection of 50mM sodium citrate solution (Sigama) 60mg/kg for 5 consecutive days.
• the model group received a single intraperitoneal injection of STZ (Sigama) 60 mg/kg for 5 consecutive days. After the injection, they could eat and drink freely. Blood glucose and body weight were measured and recorded regularly. The modeling lasted for 135 days.
• mice from each of the three groups of mice in Example 12 were injected into the tail vein with Evans blue, the retinas were dissected and separated, and vascular leakage was observed under a fluorescence microscope.
• the specific operation is as follows: 50 ⁇ L of Evans blue (Sigama) 50 mg/mL is injected into the tail vein per mouse. After blood circulation for 1.5 hours, the mice are euthanized. Both eyes are removed, fixed in 4% paraformaldehyde (Biosharp) for 45 minutes, and peeled off. The retina of the eyeball was spread out, observed under a fluorescence microscope and photographed.
• the results are shown in Figure 22.
• the retinal vascular leakage in the DME modeling group was significantly increased compared with the DME modeling control group and the blank group. There was no significant difference between the retinas of the DME modeling control group and the blank group.
• the diabetic mouse model has a DME phenotype. .
• Example 14 Inhibitory effect of XMVA09 on retinal vascular leakage in diabetic mouse model
• mice in the DME modeling group in Example 12 were injected and administered.
• the specific dosage regimen is shown in Table 6 below:
• the Evans blue tail vein injection method was used for the XMVA09 group, the control group, and the DME modeling control group in Example 12, and the retina was dissected and separated, and the vascular leakage was observed under a fluorescence microscope.
• GraphPad Prism 8.0 software was used for data processing and statistical analysis. The statistical level was set at 5% or p ⁇ 0.05, and the mean and standard error (Mean ⁇ SEM) of each analysis index were calculated. If p ⁇ 0.05, the difference was statistically significant.

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