VEGFB (vascular endothelial growth factor B) gene-carrying AAV (adeno-associated virus) vector and application thereof in preparation of medicine for treating retinitis pigmentosa

Abstract

The invention relates to the technical field of biological medicines, in particular to an AAV virus vector carrying a VEGFB (vascular endothelial growth factor B) gene and application of the AAV virus vector in preparation of a medicine for treating retinitis pigmentosa. The invention provides an AAV virus vector carrying a VEGFB gene and application of the vector in preparation of a medicine for treating retinitis pigmentosa. A VEGFB gene is combined with an AAV virus vector to construct a recombinant virus vector capable of effectively transfecting retinal cells and expressing VEGFB protein, and effective treatment on retinitis pigmentosa is realized by utilizing biological functions of oxidation resistance, apoptosis resistance, angiogenesis inhibition and the like of the VEGFB protein.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an AAV viral vector carrying the VEGFB gene and its application in the preparation of drugs for treating retinitis pigmentosa. Background Technology

[0002] Retinitis pigmentosa (RP) is a group of the most common and severe hereditary blinding retinal degenerative diseases caused by the loss or even death of photoreceptor cells and retinal pigment epithelium (RPE). It exhibits significant clinical and genetic heterogeneity, with a global incidence of 0.025%–0.04%, affecting approximately 2.5 million people, most of whom experience low vision or blindness. Typical symptoms include night blindness, decreased visual acuity, and narrowed visual field. Fundus examination reveals abnormal signs such as osteocyte-like pigmentation, retinal vascular constriction, and waxy pallor of the optic disc. Electroretinography (ERG) shows decreased amplitude even in the early stages of the disease. Disease progression is closely related to genetic factors, oxidative stress, and inflammatory responses. More than 90 related mutated genes are known, with inheritance patterns including autosomal recessive, autosomal dominant, and X-linked inheritance, with X-linked inheritance showing the most severe phenotype. Currently, there are no effective treatments available clinically, and existing interventions (such as drugs, laser therapy, surgery, gene therapy, and cell therapy) suffer from insufficient targeting and limited efficacy.

[0003] The vascular endothelial growth factor (VEGF) family plays a crucial role in angiogenesis and retinal homeostasis. VEGFB, an important member of this family, shares the VEGFR1 receptor with VEGFA and exists in two subtypes: VEGFB167 and VEGFB186. Studies have shown that VEGFB can upregulate the expression of multiple antioxidant genes, inhibit apoptosis induced by oxidative stress, and upregulate oxidation-related genes and induce retinal degeneration in VEGFB gene knockout rats. Treatment with VEGFB protein upregulates antioxidant gene expression, thereby reducing the retinal cell apoptosis rate. Research indicates that VEGFB can target and inhibit choroidal and retinal neovascularization, regulating the expression of pro-vascular survival genes through NP-1 / VEGFR-1, which is essential for retinal vascular survival. However, the specific methods, effects, and mechanisms of its in vivo intervention in retinopathy of prematurity (RP) via AAV vectors remain unclear, and systematic animal experiments are lacking.

[0004] Adeno-associated virus (AAV) is a single-stranded DNA-deficient virus with an icosahedral capsid composed of 60-mer subunits and a 4.7 kb single-stranded DNA (ssDNA) genome. Due to its high transfection efficiency, low immunogenicity, strong targeting, and cell specificity, it has become an ideal vector for RP gene therapy. However, the construction of AAV vectors targeting the VEGFB gene and their intervention effects in RP models have not yet been reported. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide an AAV viral vector carrying the VEGFB gene and its application in the preparation of drugs for treating retinitis pigmentosa.

[0006] This invention provides a highly efficient AAV viral vector carrying the VEGFB gene, with the backbone vector being pAV-CMV-P2A-GFP and the VEGFB gene being the human VEGFB-167 subtype gene.

[0007] In this embodiment of the invention, the nucleic acid sequence of the human VEGFB-167 subtype gene is shown in SEQ ID NO:1.

[0008] In this invention, the amino acid sequence encoded by the human VEGFB-167 subtype gene is shown in SEQ ID NO:3. Compared to other VEGFB subtypes, the VEGFB-167 subtype has significant advantages in treating retinitis pigmentosa. Addressing the limitations of existing technologies in RP gene therapy targets and the lack of in vivo VEGFB intervention protocols, this invention selects the VEGFB-167 subtype gene and utilizes AAV as a vector, making it easier to target and express in retinal tissue. This allows for efficient regulation of retinal vascular homeostasis and protection of photoreceptor cells and pigment epithelial cells. Experiments have demonstrated that using AAV-based VEGFB-167 subtype gene therapy can significantly improve the thickness of diseased retina, repair tissue morphology, and partially restore retinal electrophysiological function, resulting in superior therapeutic targeting and efficacy.

[0009] To improve the expression efficiency of this gene in host cells, in this invention, the 5' end of the human VEGFB-167 subtype gene is linked with a Kozak sequence. Preferably, the nucleic acid sequence of the Kozak sequence is GCCGCCACC.

[0010] To further improve the expression of the human VEGFB-167 subtype gene, the insertion site of the human VEGFB-167 subtype gene is between the Asisl site and the Mlul site of the pAV-CMV-P2A-GFP vector.

[0011] In this embodiment of the invention, the pAV-CMV-P2A-GFP vector contains a green fluorescent marker gene (GFP), the nucleic acid sequence of which is shown in SEQ ID NO:2, and the encoded amino acid sequence is shown in SEQ ID NO:4. The addition of GFP can be used for the intuitive detection of subsequent vector construction efficiency and viral transfection efficiency, and experiments have shown that the addition of GFP does not affect the physiological activity of the human VEGFB-167 subtype gene.

[0012] Furthermore, the present invention also provides a method for constructing the AAV viral vector as described above, which includes: amplifying and adding restriction enzyme sites to both ends of the human VEGFB-167 subtype gene fragment, and then ligating it with the pAV-CMV-P2A-GFP linearized plasmid digested with the same restriction endonuclease to obtain the AAV viral vector.

[0013] In this invention, after the linking, the method further includes the steps of culturing, verifying, and amplifying the AAV viral vector in Escherichia coli;

[0014] Preferably, the verified 5' sequencing primer sequence is shown in SEQ ID NO.5, and the 3' sequencing primer sequence is shown in SEQ ID NO.6.

[0015] In a specific embodiment of the present invention, the specific steps for constructing the AAV viral vector include:

[0016] Based on the nucleic acid sequence (SEQ ID NO:1) of the human VEGFB-167 subtype gene, a gene amplification primer was designed and synthesized. A Kozak sequence and an Asisl restriction site were introduced at the 5' end of the gene coding sequence, and a Mlul restriction site was introduced at the 3' end. PCR amplification was performed using this primer to obtain the human VEGFB-167 subtype gene fragment with the above sequence modifications.

[0017] The pAV-CMV-P2A-GFP vector was digested with Asisl and Mlul to obtain a linearized plasmid. The amplified human VEGFB-167 subtype gene fragment was ligated to the linearized plasmid using DNA ligase to obtain a recombinant adeno-associated virus backbone plasmid, denoted as pAV-CMV-P2A-GFP-VEGFB.

[0018] Furthermore, the construction method further includes: transforming the constructed recombinant backbone plasmid into DH5α competent cells, and after ice bath and heat shock treatment, screening positive clones on LB medium containing antibiotics; performing sequencing verification on the positive clones, wherein the 5' sequencing primer sequence is shown in SEQ ID NO:5 and the 3' sequencing primer sequence is shown in SEQ ID NO:6; and expanding the positive clones that have been correctly sequenced.

[0019] Furthermore, the present invention also provides an adeno-associated virus obtained by packaging the virus with the AAV viral vector as described above.

[0020] Accordingly, the present invention also provides a method for packaging adeno-associated virus as described above: comprising mixing the adeno-associated virus vector as described above with 293FT cells, culturing the cells and collecting the cells to obtain the adeno-associated virus.

[0021] Alternatively, the method for preparing the adeno-associated virus includes:

[0022] The VEGFB gene sequence was obtained, amplified by PCR, and ligated into an AAV vector plasmid to construct a recombinant plasmid. The recombinant plasmid and helper plasmid were co-transfected into host cells. After culture, the cells were collected, and a high-titer recombinant AAV-VEGFB vector was obtained through lysis and ultrapureization. Preferably, the host cell was a 293FT cell.

[0023] In a specific embodiment, the amount of the 293FT cells is (3~5) × 10⁻⁶. 6 The viral vector titer is not less than 1×10⁻⁶. 13 vg / mL. The carrier titer needs to reach 1×10⁻⁶. 13 A dose of at least vg / mL is required to ensure effective transduction of target cells after subretinal injection.

[0024] In a specific embodiment, the culture includes culturing the mixed cells and vector for 6 hours, then replacing the culture medium with fresh complete culture medium preheated in a 37°C water bath, and culturing for another 72 hours.

[0025] Furthermore, the present invention also provides the use of the AAV viral vector as described above and / or the adeno-associated virus as described above in the preparation of medicaments for the prevention and treatment of retinitis pigmentosa.

[0026] In this invention, the retinitis pigmentosa is retinal damage mediated by oxidative stress. More specifically, it is retinal damage mediated by oxidative stress caused by chemical drugs. The chemical drug is sodium iodate. The symptoms of retinitis pigmentosa caused by oxidative stress include, but are not limited to, degenerative changes in the retina, such as: retinal thinning, outer nuclear layer disorder, and photoreceptor cell apoptosis.

[0027] In this invention, the prevention and treatment include prevention and / or treatment. Specifically, the prevention and treatment include improving retinal structure and / or improving retinal function. More specifically, improving retinal structure includes increasing retinal thickness in the injury model, maintaining the structural integrity of the outer nuclear layer (ONL), inner nuclear layer (INL), and retinal pigment epithelium (RPE) layers, reducing RPE layer fractures (detected by OCT), and alleviating retinal cell disorder (observed by HE staining). Improving retinal function includes increasing the amplitude of a-waves and b-waves in electroretinography (ERG) (a-wave reflects photoreceptor function, and b-wave reflects bipolar cell function).

[0028] Furthermore, the present invention also provides a drug for preventing and treating retinitis pigmentosa, the drug comprising the AAV viral vector as described above and / or the adeno-associated virus as described above.

[0029] As a feasible example, the drug described in this invention also includes pharmaceutically acceptable carriers or excipients. The dosage form of the drug described in this invention includes, but is not limited to, injection solutions or suspensions for injection. Preferably, the excipient in the drug is PBS buffer.

[0030] Furthermore, the present invention also provides a method for preventing and treating retinitis pigmentosa, comprising administering the drug as described above. The administration method includes, but is not limited to, subretinal injection. Specifically, the recombinant AAV carrier is injected into the subretinal space using a microinjector, ensuring that the carrier targets retinal cells.

[0031] This invention provides an AAV viral vector carrying the VEGFB gene, and its application in the preparation of drugs for treating retinitis pigmentosa. By combining the VEGFB gene with an AAV viral vector, a recombinant viral vector capable of effectively transfecting retinal cells and expressing the VEGFB protein is constructed. Utilizing the antioxidant, anti-apoptotic, and angiogenesis-inhibiting biological functions of the VEGFB protein, effective treatment of retinitis pigmentosa can be achieved. Attached Figure Description

[0032] Figure 1 Display carrier spectrum;

[0033] Figure 2 The titer determination results are shown (Note: the sample was diluted 1000 times before the determination).

[0034] Figure 3 The results of ERG a-wave and b-wave amplitude in animal models (Note: A: empty group; B: model group; C: AAV-VEGFB group; control group).

[0035] Figure 4The results of ERG detection of retinal function a-wave and b-wave amplitudes are shown (Note: A: Model group; B: AAV-VEGFB group).

[0036] Figure 5 Statistical results of retinal thickness changes shown by OCT in rat retina (Note: A: AAV-EV group, B: model group, C: AAV-VEGFB, D: control).

[0037] Figure 6 The results of OCT retinal thickness comparison in each group are shown. Compared with the control group, the model group showed a significant difference (P < 0.001). Compared with the model group, the experimental group showed a significant difference (P < 0.01).

[0038] Figure 7 The results of HE staining of the retina are shown (Note: A: empty group; B: model group; C: AAV-VEGFB group; control group). Detailed Implementation

[0039] This invention provides an AAV viral vector carrying the VEGFB gene and its application in the preparation of drugs for treating retinitis pigmentosa. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0040] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0041] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0042] The term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist simultaneously, or B exists alone. A and B can be singular or plural.

[0043] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0044] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0045] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0046] It should be understood that in the various embodiments of this application, the sequence numbers of the above processes do not imply the order of execution. Some or all steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application. The present invention is further illustrated below with reference to embodiments:

[0047] Example 1: Construction and preparation of recombinant AAV-VEGFB vector

[0048] S1, Construction of adeno-associated virus vector

[0049] Obtain the amino acid sequence of the human VEGFB-167 isotype:

[0050] MSPLLRLLLAALLQLAPAQAPVSQPDAPGHQRKVVSWIDVYTRATCQPREVVVPLTVELMGTVAKQLVPSCVTVQRCGGCCPDDGLECVPTGQHQVRMQILMIRYPSSQLGEMSLEEHSQCECRPKKKDSAVKPDSPRPLCPRCTQHHQRPDPRTCRCRCRRRSFLRCQGRGLELNPDTCRCRKLRR (SEQ ID NO. 3)

[0051] Its encoding sequence is selected as:

[0052] ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCTAGATGGGTGCTGTCCGACATCAAGATGACCCAGTCTCCATCCTCTATGTACGCCAGCCTGGGCGAGAGTGACCATTACCTGCAAGGCCAGCCAGGACATCAACGGCTACCTGTCCTGGTTCCAGCAGAAGCCTGGCAAGTCTCCCCG GACACTGATCTACAGAGCCACAGACTGATCGACGGCGTGCCCTCCAGATTCTCCGGATCTGGATCTGGCCAGGACTACTCCCTGACCATCTCCAGCCTGGAATACGAGGATATGGGCATCTACAGCTGCCTGCAGTCCGACGACTTCCACTGACATTTGGCGCTGGCACCAAGCTGGAATTGAGA (SEQ ID NO.1)

[0053] Based on the above gene sequence, gene amplification primers were designed and synthesized. A Kozak sequence (GCCGCCACC) was added to the 5' end of the coding sequence for the VEGFB-167 subtype to improve expression efficiency, and an Asisl restriction site was added; a Mlul restriction site was added to the 3' end for ligation into the vector. The amplified gene fragment was ligated to the pAV-CMV-P2A-GFP plasmid using DNA ligase to obtain the pAV-CMV-P2A-GFP-VEGFB adeno-associated virus backbone plasmid. The plasmid carries a green fluorescent marker gene, and the nucleotide sequence of the GFP reporter gene is as follows:

[0054] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA (SEQ ID NO.2),

[0055] The amino acid sequence of green fluorescent protein is:

[0056] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLV NRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO.4).

[0057] The constructed plasmid was mixed with thawed DH5α competent cells and incubated on ice for 30 min, then incubated in a water bath at 42°C for 60 s, followed by an ice bath for 2 min. LB medium without antibiotics was added, and the mixture was shaken on a 37°C incubator for 45 min. The cells were then incubated on LB medium containing antibiotics at 37°C for 12 h, and positive clones were screened. Vector map ( Figure 1 ).

[0058] Sequencing primers:

[0059] 5' sequencing primers, gene sequence SEQ ID NO.5:

[0060] TTATTCTGAGTCCAAGCTAGGC,

[0061] 3' sequencing primers: Gene sequence SEQ ID NO.6:

[0062] TCGCCGGACACGCTGAACT.

[0063] S2, Adeno-associated virus packaging

[0064] Before packaging the virus, (3~5)×10 6 293FT cells were passaged and seeded, and cultured for 16-24 h. Following the instructions of the CPT high-efficiency transfection kit, the transfection system containing pAV-CMV-P2A-GFP-VEGFB was uniformly added to the culture dish containing the 293FT cells. The dish was gently shaken horizontally to mix, and cultured for 6 h. The culture medium was then replaced with fresh complete medium preheated to 37°C. 72 h after transfection, fluorescence was observed and photographed to determine transfection efficiency, and cells were collected. The collected cells were resuspended in PBS, and subjected to repeated freeze-thaw cycles to lyse the cells. Totipotent nuclease was added to the cell lysis buffer for half an hour, followed by induction at 15000 rpm. -1Centrifuge for 30 min and collect the supernatant. The cell lysate supernatant is then subjected to a gradient centrifugation with iodixanol to obtain preliminarily purified adeno-associated virus. Iodixanol is removed by ultrafiltration with PBS to obtain the final purified viral solution. Viral titers are calculated based on a standard curve. The main testing methods include:

[0065] Take 6 μL of purified recombinant adeno-associated virus, remove DNA contamination, and lyse the virus to obtain its genomic DNA. Then perform qRT-PCR detection, with plasmids in 10, 10... 2 10 3 10 4 and 10 5 A standard curve was prepared after dilution. The qRT-PCR reaction system was 20 μL: 10 μL of 2×Syber green mix, 0.8 μL each of forward and reverse primers, 2 μL of purified virus solution, and ddH2O to a final volume of 20 μL. The reaction conditions were 95℃ for 3 min, 95℃ for 5 s, 60℃ for 15 s, and 72℃ for 15 s, for 39 cycles. Three replicates were set up for each sample. The viral titer was calculated based on the standard curve. The results are shown below. Figure 2 In the figure, the green curve represents the sample line (diluted 1000 times), and the red line represents the standard line. The results show that the titer of the prepared AAV-VEGFB carrier is 7.28 × 10⁻⁶. 13 vg / mL

[0066] Example 2: Establishment of an animal model of retinitis pigmentosa

[0067] Experimental Methods: Forty male SD rats weighing approximately 240g were selected and divided into four groups. All rats underwent fundus examination to exclude retinal diseases. ERG examination was performed on both eyes; rats with statistically insignificant differences in a-wave and b-wave amplitude between the two eyes (P>0.05) were included, while those with significant differences were excluded. Animal models were established. In groups 1-3, a retinal damage model was initially established by intravenous injection of sodium iodate solution at a dose of 50mg / kg. One day later, subretinal viral injection was administered, with the right eye receiving 3μL of virus (suspended in PBS buffer, titer 2.18×10⁻⁶). 12 (vg / mL), the left eye was injected with an equal volume of sterile PBS solution. ERG examinations were performed on the four groups at 1 week, 4 weeks, and 6 weeks after modeling was completed, and the amplitudes of the a and b waves were counted to see if there were any statistical differences.

[0068] Experimental Results: In groups 1-3, ERG examinations after sodium iodate injection showed that the amplitudes of both a-wave and b-wave disappeared in both eyes, indicating successful modeling. In group 4, ERG examinations performed at the same time showed no difference in the amplitudes of both a-wave and b-wave compared to before PBS injection. Figure 3 ).

[0069] Example 3: Detection of the intervention effect of AAV-VEGFB on the RP model

[0070] Electroretinography (ERG) test (to assess retinal function)

[0071] Experimental method: After 12 hours of dark adaptation, mice were subjected to subsequent operations under red light. 10 g·L -1 Rats were anesthetized with sodium pentobarbital solution. Compound tropicamide eye drops were used to dilate the pupils. After applying ophthalmic gel to the cornea, the rats were placed on a temperature-controlled stage with electrodes connected. One electrode was inserted subcutaneously into the parietal bone, and the other subcutaneously into the tail. The optic disc was located under a red light field to pinpoint the rat's retina, minimizing environmental interference. The software "Visual Electrophysiology" was opened, "FERG" was selected, and channels 1 and 2 were chosen. "Settings" were then applied, with stimulation times set to 2, a frequency of 0.05 Hz, and a duration of 6 ms. A total of 5 stimulations were performed, with a 5-second interval between each stimulation. The waveform and corresponding values ​​were recorded. "Oscillography" was then selected. Once the baseline stabilized, "Acquisition" was clicked. After a "beep" sound was heard, the acquisition was complete. "Save" was then clicked.

[0072] Experimental Results: This part of the experiment aimed to investigate whether AAV-VEGFB had an improving effect on the electrophysiological function of the retina. Therefore, visual electrophysiological function was assessed by ERG at weeks 1, 4, and 6 post-injection. In the first three groups, ERG examinations at weeks 1, 4, and 6 showed no a-wave or b-wave in either eye, with no statistically significant difference (P>0.05). Similarly, in the fourth group, ERG examinations at weeks 1 and 4 also showed no a-wave or b-wave in either eye, with no statistically significant difference (P>0.05). At week 6, ERG examinations showed a-wave and b-wave in both eyes, but the amplitude of a-wave and b-wave was not statistically different from the model group (P>0.05). Figure 4 ).

[0073] Optical coherence tomography (OCT) scan (for assessing retinal structure):

[0074] Experimental Methods: After deep anesthesia, rats were mydriatic with compound tropicamide eye drops. Once the pupils were dilated, an ophthalmic gel was applied to prevent corneal dryness and damage. The rats were placed on a support, and the angle and parameters of the animal OCT imaging system were adjusted for OCT scanning. A retinal tomographic image of one optic disc diameter was collected, and the distance from the retinal ganglion cell layer to the pigment epithelium layer at this level was measured as the retinal thickness.

[0075] Experimental results: Compared with the control group (215.73±1.38) μM, the retinal thickness of the model group (162.85±9.28) μM was significantly decreased (P<0.01). The retinal thickness of the AAV-VEGFB group (188.81±12.08) μM was significantly increased compared with the model group (P<0.001). Figure 5 ).

[0076] HE staining (for assessing retinal tissue morphology)

[0077] Experimental methods: After baking the sections in an oven at 56-60°C for 2 hours, dewaxing and dehydration were performed: xylene (I) 5-10 min, xylene (II) 5-10 min, anhydrous ethanol 5 min, 90% ethanol 2 min, 80% ethanol 2 min, 70% ethanol 2 min, and distilled water 2 min. Staining: hematoxylin staining for 5-10 min, immersing in tap water to wash away excess staining solution, about 10 min, then rinsing again with distilled water (for a few seconds), differentiation for 2-30 seconds, rinsing with tap water for 10 min (differentiation solution or 5% acetic acid solution or 0.5% hydrochloric acid ethanol), eosin staining for 30 s-2 min. If direct observation is required, wash twice with 70% ethanol. If dehydration and clearing are required for mounting, 95% ethanol (I) 5 min, 70% ethanol, 80% anhydrous ethanol, 90% ethanol, and anhydrous ethanol for 10 s each, xylene (I) 5 min, and xylene (II) 5 min. Mount and fix with neutral resin, observe under an optical microscope (cell nuclei appear blue, cytoplasm appears pink or red), and acquire images.

[0078] Experimental Results: The control group's retina showed a well-structured retinal layer, including the ganglion cell layer (RGC), the inner nuclear layer (INL), and a complete inner / outer ganglion junction (IS / OS layer). The model group showed disordered arrangement of INL and ONL cells. After treatment with AAV, the pathological condition significantly improved. Figure 7 ).

[0079] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An AAV viral vector carrying a VEGFB gene, characterized in that, The backbone vector is pAV-CMV-P2A-GFP, and the VEGFB gene is a human VEGFB-167 subtype gene.

2. The AAV viral vector of claim 1, wherein, The nucleic acid sequence of the human VEGFB-167 subtype gene is shown as SEQ ID NO:

1.

3. The AAV viral vector of claim 1 or 2, wherein, The human VEGFB-167 subtype gene is connected with a Kozak sequence at the 5' end.

4. The AAV viral vector of claim 1, wherein, The insertion site of the human VEGFB-167 subtype gene is between the Asisl site and the Mlul site of the pAV-CMV-P2A-GFP vector.

5. A method of constructing an AAV viral vector according to any one of claims 1 to 4, characterised in that, The human VEGFB-167 subtype gene is amplified and enzyme cutting sites are added at both ends of the gene fragment, and then the gene fragment is connected with a linearized pAV-CMV-P2A-GFP plasmid that has been digested by the same restriction enzyme, to obtain the AAV viral vector.

6. The construction method of claim 5, wherein, The steps of culturing, verifying and expanding the AAV viral vector in E. coli are also included. The 5' end sequencing primer sequence of the verification is shown as SEQ ID NO. 5, and the 3' end sequencing primer sequence is shown as SEQ ID NO.

6.

7. An adeno-associated virus characterized in that, The AAV viral vector is packaged by the method.

8. A method of packaging an adeno-associated virus according to claim 7, wherein, The AAV viral vector is mixed with 293FT cells, and the cells are collected after culturing to obtain the AAV.

9. Use of the AAV viral vector of any one of claims 1-4 and / or the AAV of claim 7 in the preparation of a drug for preventing and treating retinal pigment degeneration.

10. A medicament for preventing and treating retinal pigment degeneration, characterized by comprising the compound according to Claim 1. The AAV viral vector of any one of claims 1-4 and / or the AAV of claim 7 are included.

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