Patent Application
20250223345
The present invention relates to the field of immunology and gene delivery. More particularly, the present application relates to compositions, systems, and methods for producing proteins of interest, such as antibodies.
Age-related macular degeneration (AMD) is a group of age-related macular diseases induced by various factors. Their common features are the lesions of the macular retina and retinal pigment epithelium and choroid, which cause visual dysfunction and progressive decrease in central vision of patients. The prevalence of AMD in China and even the world is increasing with age, and AMD is one of the main causes of irreversible vision impairment in people over 50 years old. As the population ages, AMD patients are 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, and the late stage is divided into 2 types, which are dry (atrophic) and neovascular (exudative, wet), among which, neovascular age-related macular degeneration (nAMD), also called wet age-related macular degeneration (wAMD), is a main clinical type causing vision loss, and its main feature is that choroidal neovascularization (CNV) appears in the macular area, thereby causing bleeding and exudation in the macular area. Diabetic macular edema (DME) is currently one of important causes of blindness in western developed countries, and with the improvement of living standards of people in China and the aging of the population, the prevalence rate of DME has gradually increased, which seriously affects visual function and life quality of patients.
Vascular endothelial growth factor (VEGF) is one of the most important factors in the pathogenesis of wAMD and DME, and can specifically act on vascular endothelial cells, promote the proliferation of vascular endothelial cells, induce the formation of neovessels, and increase vascular leakage. At present, it has been found that blocking the expression of VEGF can induce vascular remodeling and promote the regression of immature neovascularization. Therefore, VEGF is a promising therapeutic target for wAMD and DME. Since the application of anti-VEGF drugs represented by Ranibizumab in 2006, anti-VEGF drugs have been widely used to treat wAMD and DME. In recent 10 years, irreversible blindness caused by wAMD and DME has been greatly reduced due to the simultaneous application of multiple anti-VEGF therapies.
Although there have been great progress and breakthroughs VEGF-targeting drugs in clinicals, which can significantly reduce the degree of vascular leakage and edema, improve vision, and have not found serious complications, these protein drugs injected into the body are rapidly eliminated through metabolism, thus multiple intraocular injections are required to maintain the therapeutic effects. Long-term treatment increases the economic burden of patients, repeated injection increases the pain and the possibility of adverse reactions of patients, some degree of vision loss occurs when changing from conventional administration to low-frequency administration, and 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 art 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 present invention provides a bispecific antibody comprising anti-VEGF-A and anti-ANG-2 binding domain, which is preferably a tandem single chain antibody molecule (tandem scFv, ta-scFv), wherein the heavy chain variable regions (VH) and the light chain variable regions (VL) of the bispecific antibody are arranged from N-terminus to C-terminus in the following order:
In one embodiment, the VHanti-VEGF-A comprises a sequence as depicted in SEQ ID NO: 13 or a sequence that is 70%, 80%, 90%, 95% or 99% identical to the sequence of SEQ ID NO: 13;
In one embodiment, the antibody comprises a sequence as depicted in SEQ ID NO: 17, 18, 19, 49, 51 or 53 or a sequence that is 70%, 80%, 90%, 95% or 99% identical to the sequence of SEQ ID NO: 17, 18, 19, 49, 51 or 53.
In one embodiment, the N-terminus of the bispecific antibody construct comprises a signal peptide sequence or a tag sequence, preferably the signal peptide sequence is a CD5-sp signal peptide comprising a sequence as depicted in SEQ ID NO: 23.
In one embodiment, the antibody comprises a sequence as depicted in SEQ ID NO: 20, 21, 22, 50, 52 or 54 or a sequence that is 70%, 80%, 90%, 95% or 99% identical to the sequence of SEQ ID NO: 20, 21, 22, 50, 52 or 54.
In one embodiment, the heavy chain variable region (VH) and the light chain variable region (VL) are operably linked by (G4S)n, where n is an integer greater than 1, preferably any integer between 1 and 4, such as G4S, (G4S)2, (G4S)3 or (G4S)4, for example,
Furthermore, the present invention provides a nucleic acid sequence encoding the bispecific antibody as described above.
The present invention also provides a vector comprising a nucleic acid sequence encoding the bispecific antibody.
In one embodiment, the vector is preferably AAV virus.
In one embodiment, the AAV virus vector further comprises: a 5′ ITR and a 3′ ITR, a promoter, and a polyA sequence. In another aspect, the present invention provides an AAV virus particle, comprising any of the AAV virus vectors as described above and a capsid protein, preferably wherein the serotype of the capsid protein is AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9.
The present invention provides a pharmaceutical composition comprising at least one of the bispecific antibody, nucleic acid sequence, vector such as AAV virus vector as described above and AAV virus particle as described above, and a pharmaceutically acceptable carrier.
The present invention provides a use of the bispecific antibody, nucleic acid sequence, vector such as AAV virus vector, AAV particle as described above, or pharmaceutical compositions as described above in the preparation of a medicament for treating or preventing cancer, intraocular neovascularization syndrome, rheumatoid arthritis, psoriasis, proliferative retinopathy, age-related macular degeneration or diabetic macular edema.
In one embodiment, the age-related macular degeneration is wet age-related macular degeneration.
In a specific embodiment, the medicament is administered by intravitreal or subretinal injection.
The present invention will be described in detail below according to embodiments and in conjunction with the accompanying drawings. The above aspects of the invention and other aspects of the invention will be apparent in the following detailed description. The scope of the present invention is not limited to the following examples.
The antibodies in the present invention are multispecific, and can be humanized, single-chain, chimeric, synthetic, recombinant, heterozygous, mutant, and grafted antibodies; the antibody format in the present invention is a scFv spliced by antibody light chain variable regions (VL) and antibody heavy chain variable regions (VH), and the antibody can bind to two different antigens, such as VEGF-A and ANG-2.
The antibody light chain variable region (VL) and antibody heavy chain variable region (VH) can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), and interspersed more conserved regions called framework regions (FWR). The CDRs of the antibodies and antigen-binding fragments disclosed herein are defined or identified by Kabat numbering. In one embodiment, each VH and VL generally includes 3 CDRs and 4 FWRs arranged in the following order from the amino end to the carboxy end: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4. The CDRs of the antibodies and antigen-binding fragments disclosed herein are defined or recognized by Kabat numbering.
As used herein, “vector” refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed.
The experimental methods in the following examples, unless otherwise specified, are all conventional methods.
The VH amino acid sequence and the VL amino acid sequence of anti-VEGF, the VH amino acid sequence and the VL amino acid sequence of anti-ANG-2 were linked with (G4S)3, G4S, and (G4S)3 peptide linkers, respectively, with a secretory signal peptide CD5-sp nucleotide sequence added to the N-terminus to form an open reading frame with a structure of CD5-sp-VHanti-VEGF-A-(G4S)3-VLanti-VEGF-A-G4S-VHanti-ANG-2-(G4S)3-VLanti-ANG-2; the nucleotide sequences were designed according to human codon preference, with a BamH I cleavage site introduced at the 5′ terminus, and an EcoR V cleavage site introduced at the 3′ terminus, constructed according to the respective sequences in Table 1 and Table 2, and were named as XMVA01, XMVA04 and XMVA09 (the full-length genes were synthesized by Nanjing GenScript Biotech Corporation). The schematic diagrams of their structures are shown in
The above constructs and pAAV9neo-CAG plasmid were double digested with BamH I/EcoR V, and pAAV9-XMVA01, pAAV9-XMVA04 and pAAV9-XMVA09 vectors were constructed respectively by conventional molecular biology operations such as ligation, transformation, cloning screening and identification; the information of pAAV9-XMVA09 vector was shown in
PDDDPYYATWAKGRATIS
LDIWGQGTLVTVSS
SGVPSRFSGSGSGAEFTLT
LASTNGANFGQGTKLTVL
HWVRQAPGQGLEWMGWI
NPNSGGTNYAQKFQGRVT
DSSGYYYPGAFDIWGQGT
SSDHWVFGGGTKLTVL
GGYTYYADSVKGRFTISA
YWGQGTLVTVSS
SGVPSRFSGSGSGTDFTLT
NPWTFGQGTKVEIKR
HWVRQAPGQGLEWMGWI
NPNSGGTNYAQKFQGRVT
DSSGYYYPGAFDIWGQGT
SSSDHWVFGGGTKLTVL
The HEK293T cells were transfected with pAAV9-XMVA01, pAAV9-XMVA04 and pAAV9-XMVA09 plasmids respectively, the supernatant was collected, and the expression of proteins in the supernatant was detected by ELISA. The specific operation is as follows: 293T cells were resuspended in complete culture medium of DMEM containing 10% fetal bovine serum, inoculated into a culture dish, and cultured at 37° C. in 5% CO2 incubator until the confluence of the cells reaches 70%-90%, then the plasmid was transfected using Lipofectamine 2000 (Invitrogen) transfection reagent according to the instructions thereof. The culture was continued for 72 hours, and then the supernatant was collected and stored at −80° C. for later use.
The specific detection method of ELISA was as follows: an ELISA plate (Thermo) was coated with 0.1 μg/mL VEGF165 (proteintech) protein, 100 μL per well, incubated overnight at 4° C., and the ELISA plate was washed three times with PBS containing 0.05% Tween (Sangon) for 3 minutes each time. The plates were blocked with PBS 200 μL/well containing 2% BSA (MikeBio) for 1 hour at room temperature and washed three times again. Conbercept (10 mg/mL) standard and the cell supernatants collected after transfecting HEK293T cells with pAAV9-XMVA01, pAAV9-XMVA04, and pAAV9-XMVA09 plasmids were added to the ELISA plate. The standard was diluted at 8 continuous dilutions starting at 312.5 ng/mL from the first well, and the supernatants to be tested were diluted at 1:10 or 1:50, the data were averaged, and a cell supernatant without plasmid transfection was used as a Control group, and the plate was incubated at 37° C. for 1 hour, and washed three times. A 1:5000 dilutions of goat anti-human IgG antibody (Jackson Immuno Research) labeled with horseradish peroxidase was added to each well, and the plate was incubated at 37° C. for 1 hour, and then washed three times. 100 μL/well of TMB chromogen solution (Beyotime) was added to each well, and reacted for 6 minutes at room temperature in the dark, and 100 μL/well of stop solution (Beyotime) was added to the above wells to terminate the reaction. A microplate reader was used to detect the OD value at 450 nm, and the contents of the sample to be tested were calculated. The results are shown in
The cell density of HRMECs was adjusted to 4×104/mL with complete culture medium (Science Cell) of the ECM containing 1% ECGS, and the cells were inoculated 100 μL/well into a flat-bottom 96-well plate; after the pAAV9-XMVA01, pAAV9-XMVA04 and pAAV9-XMVA09 plasmids were transfected into HEK293T cells, respectively, the collected cell supernatants were added into the wells at 100 μL/well, and the cell supernatant without transfected plasmids was used as a Control group, and the cells were cultured for 72 hours at 37° C. in a 5% CO2 incubator. 20 μL/well of CCK-8 solution was added to the experimental group and control group respectively according to the instructions of Cell Counting Kit-8 (CCK-8, Biosharp). After incubation for 4 hours, a microplate reader was used to detect the OD value at 450 nm wavelength. The results are shown in
The thawed matrigel (Corning) was uniformly spreaded in a flat-bottom 96-well plate, 50 μL/well, and then the plate was incubated at 37° C. for 1 hour in a 5% CO2 incubator; after the pAAV9-XMVA01, pAAV9-XMVA04 and pAAV9-XMVA09 plasmids were transfected into HEK293T cells, respectively, the collected cell supernatants were used to treat HRMECs, the cell supernatant without transfected plasmids was used as a control group, and the cells were resuspended with a basal medium of ECM without growth factors and serum after cultured for 48 hours, adjusted the cell density to 2×105/mL, and inoculated at 100 μL/well in a 96-well plate containing matrigel, and cultured in a 5% CO2 incubator at 37° C., and observed once every 2 hours; after 4 to 8 hours, the cells were observed and photographed under a microscope, and the influence of the supernatants to be tested on the formation of HRMECs tubes were recorded. The results are shown in
The VH amino acid sequence and the VL amino acid sequence of anti-VEGF, the VH amino acid sequence and the VL amino acid sequence of anti-ANG-2 were linked with G4S, (G4S)3 and G4S peptide linkers, respectively, with a secretory signal peptide CD5-sp nucleotide sequence added to the N-terminus to form an open reading frame with the structure of CD5-sp-VLanti-ANG-2-G4S-VHanti-VEGF-A-(G4S)3-VLanti-VEGF-A-G4S-VHanti-ANG-2; the nucleotide sequence was designed according to human codon preference, with a BamH I cleavage site introduced at the 5′ terminus, and an EcoR V cleavage site introduced at the 3′ terminus, and was named as XMVA10 (
The VH amino acid sequence and the VL amino acid sequence of anti-VEGF, the VH amino acid sequence and the VL amino acid sequence of anti-ANG-2 were linked with G4S, (G4S)3 and G4S peptide linkers, respectively, with a secretory signal peptide CD5-sp nucleotide sequence added to the N-terminus to form an open reading frame with the structure of CD5-sp-VLanti-VEGF-A-G4S-VHanti-ANG-2-(G4S)3-VLanti-ANG-2-G4S-VHanti-VEGF-A; the nucleotide sequence was designed according to human codon preference, with a the BamH I cleavage site introduced at the 5′ terminus, and an EcoR V cleavage site introduced at the 3′ terminus, and was named as XMVA11 (
The VH amino acid sequence and the VL amino acid sequence of anti-VEGF, the VH amino acid sequence and the VL amino acid sequence of anti-ANG-2 were linked with (G(S)3, G4S and (G4S)3 peptide linkers, respectively, with a secretory signal peptide CD5-sp nucleotide sequence added to the N-terminus to form an open reading frame with the structure CD5-sp-VHanti-VEGF-A-(G4S)3-VLanti-VEGF-A-G4S-VHanti-ANG-2-(G4S)3-VLanti-ANG-2; the nucleotide sequence was designed according to human codon preference, with a BamH I cleavage site introduced at the 5′ terminus, and an EcoR. V cleavage site introduced at the 3′ terminus, and was named as XMVA13 (
The VH amino acid sequence and the VL amino acid sequence of anti-VEGF, the VH amino acid sequence and the VL amino acid sequence of anti-ANG-2 were linked with G4S, (G4S)3 and G4S peptide linkers, respectively, with a secretory signal peptide CD5-sp nucleotide sequence added to the N-terminus to form an open reading frame with the structure of CD5-sp-VLanti-ANG-2-G4S-VHanti-VEGF-A-(G4S)3-VL anti-VEGF-A-G4S-VHanti-ANG-2; the nucleotide sequence was designed according to human codon preference, with a BamH I cleavage site introduced at the 5′ terminus, and an EcoR V cleavage site introduced at the 3′ terminus, and was named as XMVA14 (
The VH amino acid sequence and the VL amino acid sequence of anti-VEGF, the VH amino acid sequence and the VL amino acid sequence of anti-ANG-2 were linked with G4S, (G4S)3 and G4S peptide linkers, respectively, with a secretory signal peptide CD5-sp nucleotide sequence added to the N-terminus to form an open reading frame with the structure of CD5-sp-VLanti-VEGF-A-G4S-VHanti-ANG-2-(G4S)3-VLanti-ANG-2-G4S-VHanti-VEGF-A; the nucleotide sequence was designed according to human codon preference, with a BamH I cleavage site introduced at the 5′ terminus, and an EcoR V cleavage site introduced at the 3′ terminus, and was named as XMVA15 (
XMVA10, XMVA11, XMVA13, XMVA14 and XMVA15 vectors were constructed through conventional molecular biology operations such as ligation, transformation, cloning screening and identification, and high-quality plasmid DNA was obtained for later use by using an endotoxin-free plasmid extraction kit (MN).
According to the method described in Example 2, plasmids were transfected into HEK293T cells, respectively, supernatants were collected, and expression of protein in supernatant was detected by ELISA. The effect of expressing the above vectors in cells on the proliferation of HRMECs was detected according to the method described in Example 3. The effect of expressing the above plasmids in cells on HRMECs tube formation was detected according to the method described in Example 4. Recombinant AAV viruses were prepared and identified according to the method described in Example 5.
The XMVA09 construct and ssAAV plasmid were double digested with BamH I/EcoR V, and ssAAV-XMVA09 vector was constructed by conventional molecular biology operations such as ligation, transformation, cloning screening and identification, and the vector information is shown in
model at home and abroad, and is a standard animal model in most treatment evaluation experiments at present. It selectively destroys the photoreceptor outer segment disc membrane, Bruch membrane, retinal pigment epithelium and part of the anterior choroidal capillary network are selectively damaged, followed by a damage repair reaction, including invasion and growth of fibroblast, retinal pigment epithelial cell and vascular endothelial cell, and finally, neovascularization is formed in a photocoagulation area.
Thirty SPF grade 6˜8 weeks male C57 BL/6J mice (purchased from Beijing Vital River Laboratories Experimental Animal Technology Co., Ltd.), weighing 22-25 g were used, and the specific administration scheme is shown in Table 4 below:
Since the expression of the target gene reached a stable level after a certain period of time after AA V injection, intravitreal injection was performed on day 1, and binocular funduslaser photocoagulation was performed on the successfully injected animals on day 22 to induce CNV model.
Establishment of mouse CNV model: The pupils of both eyes of the mice were dilated with 1-2 drops of topicamide eye drops, and 5% chloral hydrate was injected intramuscularly for anesthesia. After anesthesia, carbomer eye drops were dropped in both eyes, a fundus laser scope was placed, and photocoagulation was performed around the optic papilla at a distance of about 1.5-2 PD from the optic disc avoiding blood vessels. Laser parameters were as follows: wavelength 532 nm, energy 80 mW, spot size 50 μm, exposure time 100 ms, and erythromycin eye ointement were applied to both eyes of the animal after photocoagulation.
On day 29, fluorescein fundus angiography (FFA) detection was performed on mice: fluorescein sodium injection (15 mg/mL, 10 mL/kg) was intraperitoneally injected, several clear pictures of both eyes were collected at early (within 1.5 minutes) and late (after 3 minutes) stages after the fluorescein sodium injection, the fluorescence leakage degree of the effective light spots were rated, and the percentage of grade 3 leakage light spots and the mean score of leakage light spots were calculated. The results are shown in
Effective light spot refers to a light spot that has no severe retinal hemorrhage nearby and can be completely displayed in the FFA. The grading standards of spots fluorescence leakage are as follows: grade 0 (no fluorescence leakage), grade 1 (mild fluorescence leakage, with a leakage area of 1-50% of the laser spot size), grade 2 (moderate fluorescence leakage, with a leakage area of 50-100% of the laser spot size), grade 3 (severe fluorescence leakage, with a leakage area larger than the laser spot size).
Percentage (%) of light spots of each grade=total number of light spots of the corresponding grade÷total number of 4 types of light spots×100%.
Mean score of leakage light spot=[(number of grade 0 light spots×0)+(number of grade 1 light spots×1)+(number of grade 2 light spots×2)+(number of grade 3 light spots×3)]÷total number of 4 types of light spots.
Seven male rhesus monkeys (purchased from Ya'an Primed Bio-tech Co., Ltd.) with a common grade (qualified in quarantine before test) of 5-6 years old were used, and the specific administration scheme is shown in table 5 below:
XMVA09 Group: AAV-XMVA09 (50 L/eye) was administered by intravitreal injection in both eyes on day 21 before laser modeling, and CNV was induced in both eyes by fundus laser on days 0.
Control group: CNV was induced in both eyes by fundus laser on day 0, and PBS (50 μL/eye) was administered by intravitreal injection in both eyes on day 21.
All animals were examined by optical coherence tomography (OCT) prior to laser modeling and fundus photography (FP), FFA and OCT examinations were performed on day 19 and day 77 of laser modeling; and the number, leakage area and retinal thickness of grade IV light spots were used as main efficacy evaluation indicators. Establishment of the rhesus monkey CNV model: animals were anesthetized by intramuscular injection of ketamine hydrochloride (20 mg/kg) and dexmedetomidine hydrochloride (0.03 mg/kg), and the pupils of both eyes were dilated with Midori (compound tropicamide eye drops) drops. After the pupils of both eyes were larger than 6 mm, the head of the animal was fixed in front of an ophthalmic laser photocoagulator, and after the retinal structure was peculated through a fundus scope, laser photocoagulating was carried out at an optic disc distance around the center of the macula, with 9 points in each eye. The laser parameters were: wavelength 532 nm, energy 650 mW-700 mW, spot size 50 μm, exposure time 0.1 second. Fluorescein leakage area was measured by FP and FFA, and the specific examination method was as follows: (1) animals were anesthetized by intramuscular injection of ketamine hydrochloride (20 mg/kg) and dexmedetomidine hydrochloride (0.03 mg/kg). (2) The pupils of both eyes were dilated with 1-2 drops of compound tropicamide eye drops. (3) The pupil of the animal was observed after pupil dilation, if the pupil was greater than 6 mm under light, a colour photograph of the fundus centered on the macula was taken by a fundus camera. (4) After that, a 0.075 mL/kg dose of 10% fluorescein sodium injection (Fluorescite, Alcon) was rapidly injected from the saphenous vein of the lower limb. FFA photographs centered on the macula were taken by fundus cameras at early (within 1 minute), medium (5 minutes) and late (10 minutes) stages after fluorescein injection. (6) The fluorescein leakage area of the grade IV spot in the late stage (10 min) of FFA photo was automatically measured by Image J software (version 1.53e, NIH, Wayne, Raband, USA) as an evaluation indicator of the efficacy of FFA. The fluorescein leakage grading standard of the laser spot is: grade I (the light spot has no high fluorescence), grade II (the light spot has high fluorescence without leakage), grade III (the light spot has high fluorescence with slight fluorescein leakage, but leakage does not exceed the edge of the light spot), grade IV (the light spot has high fluorescence with significant fluorescein leakage, and leakage exceeds the edge of the light spot).
Retinal thickness changes were measured by OCT, and the specific examination method is as follows: (1) after completing the FP and FFA examination, the animals were placed in front of an OCT instrument, and the animal's eyes were adjusted to look directly at the scanning lens. (2) Multi-layer linear scanning was performed by adopting a follow-up mode centered on the macula, with the scanning area covered all laser points. (3) A layer with the most obvious retinal thickness change and clear retinal boundary after modeling was selected, and the maximum retinal thickness value of the region was measured by using measurement software carried by the instrument to serve as an OCT efficacy evaluation indicator.
The results are shown in
The 24-well plate was pre-treated with 0.1% gelatin at 37° C. for 1-2 bouts and then dried for several minutes; then the HRMECs were resuspended in complete culture medium of ECM (Sciencell) containing 10% FBS+1% PS+1% ECGS, and spread evenly in the well plate according to the standard of 3×105/well; on the next day, starvation treatment was initiated with a low-serum culture medium containing 0.5% FBS for 7 hours; then the HRMECs were exposed to 30 mM high glucose environment overnight; the control group was not treated with high glucose, on the next day, immunofluorescence co-staining of cells for membrane protein with VE-cadherin and nuclear with DAPI was performed, for fluorescence microscope observation and photography. The results are shown in
Steps of immunofluorescence staining: discarding the supernatant of the culture medium so that the remaining volume was 200 μL, and adding 200 μL of 4% PFA to start the gradient fixation for 20 minutes at room temperature; after discarding the supernatant, directly adding 200 μL of PFA, and continuing to fix at room temperature for 10 minutes; after discarding the fixation solution, washing with PBS for 3 times, each time for 5 minutes; diluting VE-cadherin (Santa Cruz Biotechnology Inc) at 1:150 with PBS, then adding 300 μL/well, and incubating at room temperature for 2 hours; discarding the supernatant, washing with PBS for 3 times, each time for 5 minutes; diluting the Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (Invitrogen) at 1:200 with PBS, adding 200 μL per well, and incubating at 37° C. for 1 hour; discarding the supernatant, washing with PBS for 3 times, each time for 5 minutes; and finally adding DAPI-containing mounting agent (Beyotime), 100 μL/well, and observing and photographing under a fluorescence microscope.
The 24-well plate was pre-coated with 0.1% gelatin at 4° C. overnight or at 37° C. for 2 hours; the coating solution was discarded, and HRMECs cells were inoculated 3×105/well; on the next day, when the monolayer cells reached a certain confluence, starvation treatment was initiated with a low-serum medium containing 0.5% FBS for 7 hours; the supernatant of untransfected HEK293T cells and the supernatant of HEK293T cells transfected with ssAAV-XMVA09 plasmid were co-incubated with high glucose (30 mM glucose), respectively. HRMECs were treated overnight, and the control group was a non-modeling group without high glucose treatment. Immunofluorescence staining of VE-cadherin was performed, and the detailed steps were as in Example 9, and photographic analysis was performed under a fluorescence microscope. The results are shown in
Gelatin was treated with EZ-Link™ NHS-LC-LC-biotin to obtain biotinylated gelatin; the biotinylated gelatin was used to coat a cell culture plate at 4° C. overnight of 37° C. for 2 hours; HRMECs were directly inoculated in the culture plate, and the inoculation amount of cells was 3×105/well; on the next day, starvation treatment was first initiated with a low-serum medium containing 0.5% FBS for 7 hours; then the supernatant of untransfected HEK293T cells and the supernatant of HEK293T cells transfected with ssAAV-XMVA09 plasmid were co-incubated with high glucose (30 mM glucose), respectively, HRMECs were treated for 17 hours, and the control group was a non-modeling group without high glucose treatment. After staining with FITC-avidin and fixation with 4% PFA, staining with AF594-phaloidin (Invitrogen A12381) was started, then an anti-fluorescence quencher containing DAPI was added, and photographical analysis was performed under a fluorescence microscope. The results are shown in
Drug induction is one of the current methods for establishing diabetic animal models, which can better simulate clinical pathological manifestations and clinical characteristics of diabetes. Streptozotocin (STZ) is the most commonly used medicament for inducing diabetes, and it mainly causes hyperglycemia by destroying pancreatic beta cells, which in turn causes pericapillary cell loss, vascular layer thinning and blood-retinal barrier destruction, further causes vessel leakage, and it is a good model for studying DME.
Eighty SPF grade 6˜8 weeks male C57 BL/6J mice (purchased from Zhejiang Vital River Laboratories Experimental Animal Technology Co., Ltd.), weighing 20-25 g, were randomly divided into blank group, DME model control group, and DME modeling group. Before modeling, fasting blood glucose and body weight of three groups of mice were measured; after fasting for 6 hours (with free access to water), blood was collected at the tail tip for blood glucose measurement and the mice were weighed, on day 0 of modeling. Modeling was started on day 1, and mice were fasted (with free access to water) for 6 hours. The Blank group was not treated, the DME model control group recieved a single intraperitoneal injection of 50 mM sodium citrate solution (Sigma) 60 mg/kg for 5 continuous days, and the DME model group recieved a single intraperitoneal injection of STZ (Sigma) 60 mg/kg for 5 continuous days. The mice were free to eat and drink, blood glucose and body weight were measured and recorded regularly, and the modeling lasted for 135 days.
Results are shown in
On day 81 after modeling, a portion of mice in each of the three groups of mice in Example 12 were injected with Evans Blue by tail vein injection, the retinas were dissected and separated, and vascular leakage was observed under a fluorescence microscope. The specific operations are as follows: 50 mg/mL of Evans blue (Sigma) was injected 50 μL/mouse into the tail vein; after 1.5 hours of blood circulation, the mice were euthanized, both eyes were enucleated and fixed for 45 minutes in 4% paraformaldehyde (Biosharp), the retina of the eyeball was peeled off and spread, and observed and photographed under a fluorescence microscope.
Results are shown in
The DME model mice in Example 12 were administered by injection on day 95 after molding, and the specific administration scheme is shown in Table 6 below:
On day 29 after injection, mice in the above XMVA09 group, Control group and mice in the DME model control group in Example 12 were injected with Evans blue by tail vein injection and dissect the retina to observe blood vessel leakage under fluorescence microscope.
The results are shown in
GraphPad Prism 8.0 software was used for data processing and statistical analysis. The statistical level was set to 5% or p≤0.05, and the average number and standard error (Mean±SEM) of each analysis indicator were calculated, where p≤0.05 means the difference is statistically significant.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202210356939.6 | Apr 2022 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2023/085610 | 3/31/2023 | WO |
