GRNA, AAV expression vector and CRISPR / Cas9 system for treating corneal neovascular diseases

Abstract

The invention provides a gRNA, an AAV expression vector and a CRISPR / Cas9 system for treating corneal neovascular diseases, and particularly provides a composition which comprises: (a) a gene editing protein or an expression vector thereof, the gene editing protein comprising type II Cas protein; the gene editing protein comprises (a) a gene editing protein and (b) gRNA or an expression vector thereof, the gRNA guides the gene editing protein to be specifically combined with DNA of a VEGFA gene, a gRNA targeting area is an exon 1 of a VEGFA gene sequence, and the combination of the gRNA specifically targeting the exon 1 of the VEGFA gene sequence and the gene editing protein can effectively prevent and / or treat corneal neovascularization diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine. Specifically, the present invention relates to a gRNA, an AAV expression vector, and a CRISPR / Cas9 system for treating corneal neovascular diseases. Background Art

[0002] Corneal neovascularization (CNV) is common worldwide. Many severe cases ultimately require keratoplasty to restore vision. However, CNV is also a risk factor and major complication of corneal transplantation failure. Among the diseased corneas removed by keratoplasty, samples containing CNV account for up to 20%. The cornea is an important refractive structure in the visual system, and the avascularity of the cornea is essential to maintaining corneal transparency. Under normal circumstances, the cornea is an immune-privileged organ without blood and lymphatic vessels. However, under the influence of pathogenic factors such as infection, allergy, trauma, immune rejection, ischemia, hypoxia, and chemical damage, the limbal vascular network will form new capillaries. New blood vessels invade the cornea, resulting in corneal stroma opacity, corneal surface irregularities, corneal scarring and edema, lipid deposition, light blockage, and vision loss.

[0003] At present, clinical treatments for CNV include corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), photodynamic therapy (PDT), laser photocoagulation, fine needle diathermy, and amniotic membrane transplantation, but these therapies have limited effects, short duration of action (requires multiple doses), and some serious side effects. For example, corticosteroids can cause high intraocular pressure, cataracts, and superinfection, NSAIDs can cause corneal epithelial damage and delayed epithelial wound healing, laser photocoagulation treatment is prone to recurrence, collagen contraction and damage to the adjacent stroma of the diathermy site, changes in corneal curvature, and even stimulation of further CNV formation. The anti-angiogenic factors and pro-angiogenic factors of a normal healthy cornea maintain a stable balance. Once the factors that promote angiogenesis are greater than the factors that inhibit angiogenesis, pathological new blood vessels will form in the cornea. Vascular endothelial growth factor (VEGF) is one of the strongest angiogenic factors. The upregulated expression of vascular endothelial growth factor receptor A (VEGFA) in its family is the most important cause of CNV. Therefore, VEGFA is a therapeutic target for corneal neovascularization. Inhibitory antibodies targeting the VE GFA signaling pathway have shown good results in the treatment of CNV, but current anti-VE GFA treatment is generally only effective for active blood vessels, and the effect is short-lived and incomplete, requiring repeated injections, CNV may recur, and side effects such as corneal thinning may occur. Therefore, the clinical treatment of CNV remains a huge challenge.

[0004] The unique anatomical and immune characteristics of the cornea make it an ideal site for gene therapy. Using gene silencing technology to downregulate angiogenic genes is an emerging strategy for treating CNV, which is expected to solve the difficulties faced by traditional treatment methods. Some studies have made relevant attempts in gene therapy for CNV, and have shown excellent potential in animal studies. For example, subconjunctival injection of recombinant adenovirus vectors (rAAV) to deliver miR-204 into the damaged cornea prevents CNV generation; rAAV transfection of corneal epithelial cells with anti-VEGFA antibodies partially inhibits CNV in mouse models, but high-dose rAAV vector injection induces strong corneal immune response and inflammation. The above effects rely on early and frequent administration, and the safety issues caused by overexpression of vectors and transgenes limit their application.

[0005] As a cutting-edge gene editing technology for manipulating the genome, clustered regularly interspaced shortpalindromic repeats (CRISPR) has great application prospects in the biomedical field. CRISPR / Cas9 is based on the Cas9 endonuclease guided by CRISPR-related RNA. It searches for paired sequences on the genome through a short guide RNA (gRNA), guides Cas9 to a specific location in a complex genome, and achieves simple, rapid and precise editing of the mammalian genome. Because it can directly edit genomic DNA, the resulting effect of inhibiting gene expression is long-lasting and stable.

[0006] However, there is currently a lack of a dual AAV vector system that uses the CRISPR / Cas9 system carrying efficient gRNA to inhibit VEGFA gene expression and effectively intervene in corneal neovascularization animal models.

[0007] Therefore, there is an urgent need in the art to develop a gRNA that can effectively inhibit the expression of the VEGFA gene, and can deliver the CRISPR / Cas9 system and gRNA that play a gene editing role into the animal body through a dual AAV expression vector system to edit the VEGFA gene, and provide a new application of the system for preparing gene therapy drugs for the treatment of corneal neovascularization diseases. Summary of the invention

[0008] The purpose of the present invention is to provide a gRNA that can effectively inhibit the expression of the VEGFA gene, and can deliver the CRISPR / Cas9 system and gRNA that play a gene editing role into the animal body through a dual AAV expression vector system to play a role in editing the VEGFA gene, and provide a new application of the system for preparing gene therapy drugs for treating corneal neovascularization diseases.

[0009] The first aspect of the present invention provides a composition comprising:

[0010] (a) a gene editing protein or an expression vector thereof, wherein the gene editing protein comprises a type II Cas protein; and

[0011] (b) gRNA or its expression vector, wherein the gRNA guides the gene editing protein to specifically bind to the DNA of the VEGFA gene, and the region targeted by the gRNA is the 5'UTR region, exon 1, exon 6, and 3'UTR region of the VEGFA gene sequence.

[0012] In another preferred embodiment, the region targeted by the gRNA is positions 14538-14557, and / or positions 14377-14396, and / or positions 10589-10608, and / or positions 14370-14389, and / or positions 454-473, and / or positions 493-512, and / or positions 1095-1114 of the VEGFA gene sequence (Gene ID: 7422).

[0013] In another preferred embodiment, the gene editing protein includes Cas9.

[0014] In another preferred embodiment, the source of the Cas9 is selected from the following group: Ruminococcus Flavefaciens, Streptococcus pyogenes, Staphylococcus aureus, Acidaminococcus sp, Lachnospiraceae bacterium, or a combination thereof.

[0015] In another preferred embodiment, the nucleotide sequence of the gRNA is selected from the following group: SEQ ID NO.: 1, 2, 3, 4, 5, 6 and 7.

[0016] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0017] In another preferred embodiment, the composition further comprises:

[0018] (c) Other drugs for the prevention and / or treatment of corneal neovascularization diseases.

[0019] In another preferred embodiment, other drugs for preventing and / or treating corneal neovascularization diseases are selected from the following group: Bevacizumab, Pegaptanib, Ranibizumab, Aflibercept, Aganirsen, or a combination thereof.

[0020] In another preferred embodiment, the corneal neovascularization disease is selected from the following group: herpes simplex virus keratitis, herpes zoster keratitis, bacterial keratitis, syphilitic interstitial keratitis, trachomatous keratoconjunctivitis, fungal keratitis, ocular onchocerciasis, Acanthamoeba keratitis, corneal transplant rejection, graft-versus-host disease, specific keratoconjunctivitis, ocular rosacea, ocular cicatricial (mucosal) pemphigoid, congenital epidermal dysplasia, Turner syndrome, chemical damage (especially corneal alkali burns), thermal damage, ultraviolet keratitis, primary limbal stem cell deficiency, pterygium, corneal contact lens keratitis, mechanical corneal trauma, non-infectious corneal ulcer, corneal foreign body (such as sutures, intracorneal rings), intraepithelial neoplasia and squamous cell carcinoma, or a combination thereof.

[0021] In another preferred embodiment, the expression vector comprises a viral vector.

[0022] In another preferred embodiment, the viral vector is selected from the following group: adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes virus, SV40, poxvirus, or a combination thereof.

[0023] In another preferred embodiment, the vector is selected from the following group: lentivirus, adenovirus, adeno-associated virus (AAV), or a combination thereof. Preferably, the vector is adeno-associated virus (AAV).

[0024] In another preferred embodiment, the vector comprises AAV2 or AAV9.

[0025] In another preferred example, the expression vector of the gene editing protein and the expression vector of the gRNA are the same vector or different vectors.

[0026] In another preferred embodiment, the gene encoding the gene editing protein and the gRNA are located in the same expression vector or different expression vectors (such as AAV vector).

[0027] In another preferred embodiment, the dosage form of the composition is selected from the following group: a lyophilized preparation, a liquid preparation, or a combination thereof.

[0028] In another preferred embodiment, the composition is in the form of a liquid preparation.

[0029] In another preferred embodiment, the composition is in the form of an injection.

[0030] In another preferred embodiment, the composition is a cell preparation.

[0031] In another preferred embodiment, the weight ratio of component (a) to component (b) is 100:1-0.01:1, preferably, 10:1-0.1:1, and more preferably, 2:1-0.5:1.

[0032] In another preferred embodiment, in the composition, the content of component (a) is 0.001%-99%, preferably, 0.1%-90%, more preferably, 1%-70%.

[0033] In another preferred embodiment, in the composition, the content of component (b) is 0.001%-99%, preferably, 0.1%-90%, more preferably, 1%-70%.

[0034] In another preferred embodiment, in the composition, the content of component (c) is 1%-99%, preferably, 10%-90%, more preferably, 30%-70%.

[0035] In another preferred embodiment, in the composition, the component (a), component (b) and optional component (c) account for 0.01-99.99 wt %, preferably 0.1-90 wt %, and more preferably 1-80 wt % of the total weight of the composition.

[0036] A second aspect of the present invention provides a medicine kit, comprising:

[0037] (a1) a first container, and a gene editing protein or its expression vector, or a drug containing the gene editing protein or its expression vector, located in the first container, wherein the gene editing protein comprises a type II Cas protein;

[0038] (b1) a second container, and a gRNA or its expression vector located in the second container, or a drug containing the gRNA or its expression vector, wherein the gRNA guides the gene editing protein to specifically bind to the DNA of the VEGF A gene, and the region targeted by the gRNA is the 5'UTR region, exon 1, exon 6, and 3'UTR region of the VEGFA gene sequence.

[0039] In another preferred embodiment, the region targeted by the gRNA is positions 14538-14557, and / or positions 14377-14396, and / or positions 10589-10608, and / or positions 14370-14389, and / or positions 454-473, and / or positions 493-512, and / or positions 1095-1114 of the VEGFA gene sequence (Gene ID: 7422).

[0040] In another preferred embodiment, the gene editing protein includes Cas9.

[0041] In another preferred embodiment, the source of the Cas9 is selected from the following group: Ruminococcus Flavefaciens, Streptococcus pyogenes, Staphylococcus aureus, Acidaminococcus sp, Lachnospiraceae bacterium, or a combination thereof.

[0042] In another preferred embodiment, the nucleotide sequence of the gRNA is selected from the following group: SEQ ID NO.: 1, 2, 3, 4, 5, 6 and 7.

[0043] In another preferred embodiment, the medicine kit further comprises:

[0044] (c1) a third container, and other drugs for preventing and / or treating corneal neovascularization diseases in the third container.

[0045] In another preferred embodiment, the first container, the second container and the third container are the same or different containers.

[0046] In another preferred embodiment, the drug in the first container is a single preparation containing a gene editing protein or an expression vector thereof.

[0047] In another preferred embodiment, the drug in the second container is a single preparation containing gRNA or its expression vector.

[0048] In another preferred embodiment, the drug in the third container is a single-ingredient preparation containing other drugs for preventing and / or treating corneal neovascularization diseases.

[0049] In another preferred embodiment, the dosage form of the drug is selected from the following group: a lyophilized preparation, a liquid preparation, or a combination thereof.

[0050] In another preferred embodiment, the dosage form of the drug is an injection form.

[0051] In another preferred embodiment, the kit further contains instructions.

[0052] The third aspect of the present invention provides a use of the composition described in the first aspect of the present invention or the medicine kit described in the second aspect of the present invention for preparing a medicament for preventing and / or treating corneal neovascularization disease.

[0053] In another preferred example, in the composition, the effective concentration of the gene editing protein or its expression vector is 1.0E+7vg / μl-1.0E+10vg / μl.

[0054] In another preferred example, in the composition, the effective concentration of the gRNA or its expression vector is 1.0E+7vg / μl-1.0E+10vg / μl.

[0055] In another preferred embodiment, in the pharmaceutical composition, the effective concentration of the other drugs for preventing and / or treating corneal neovascularization disease is 0.1 μg / μl-100.0 μg / μl.

[0056] In another preferred embodiment, the composition or kit comprises (a) a gene editing protein or an expression vector thereof; and (b) a gRNA or an expression vector thereof; and (c) optionally other drugs for preventing and / or treating corneal neovascularization diseases; and (d) a pharmaceutically acceptable carrier.

[0057] In another preferred embodiment, in the composition or medicine kit, (a) gene editing protein or its expression vector; and (b) gRNA or its expression vector; and (c) optional other drugs for preventing and / or treating corneal neovascularization diseases account for 0.01-99.99wt%, preferably 0.1-90wt%, and more preferably 1-80wt% of the total weight of the composition or medicine kit.

[0058] A fourth aspect of the present invention provides a method for preventing and / or treating corneal neovascularization disease, comprising:

[0059] The composition of the first aspect of the present invention or the kit of medicine of the second aspect of the present invention is administered to a subject in need thereof.

[0060] In another preferred embodiment, the subject includes a human or non-human mammal suffering from corneal neovascularization disease.

[0061] In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

[0062] A fifth aspect of the present invention provides an AAV vector, comprising:

[0063] (a) a coding sequence of a gene editing protein, wherein the gene editing protein comprises a type II Cas protein; and

[0064] (b) gRNA, wherein the gRNA guides the gene editing protein to specifically bind to the DNA of the VEGFA gene, and the region targeted by the gRNA is the 5'UTR region, exon 1, exon 6, and 3'UTR region of the VEGFA gene sequence.

[0065] In another preferred embodiment, the region targeted by the gRNA is positions 14538-14557, and / or positions 14377-14396, and / or positions 10589-10608, and / or positions 14370-14389, and / or positions 454-473, and / or positions 493-512, and / or positions 1095-1114 of the VEGFA gene sequence (Gene ID: 7422).

[0066] In another preferred embodiment, the gene editing protein includes Cas9.

[0067] In another preferred embodiment, the source of the Cas9 is selected from the following group: Ruminococcus Flavefaciens, Streptococcus pyogenes, Staphylococcus aureus, Acidaminococcus sp, Lachnospiraceae bacterium, or a combination thereof.

[0068] In another preferred embodiment, the nucleotide sequence of the gRNA is selected from the following group: SEQ ID NO.: 1, 2, 3, 4, 5, 6 and 7.

[0069] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 It shows that the gRNA guides the gene editing protein to specifically bind to the DNA site of the VEGFA gene.

[0071] Figure 2 A CRISPR / Cas9 backbone plasmid map is shown.

[0072] Figure 3 Editing of VEGFA by different gRNAs in T7EI experiments is shown.

[0073] Figure 4 The ELISA experiment is shown to detect the inhibition of VEGFA gene expression in cell supernatant by different gRNAs.

[0074] Figure 5 Sanger sequencing was used to detect the editing effects of different gRNAs on the VEGFA gene.

[0075] Figure 6 Western blotting experiments show the inhibition of VEGFA protein expression by different gRNAs.

[0076] Figure 7 Gray value quantitative analysis of Western blotting experiments is shown.

[0077] Figure 8 The quantitative results of off-target site detection and grayscale analysis predicted by T7EI for gRNA1, gRNA2, and gRNA3 are shown.

[0078] Fig. 9Confocal microscopy is shown to detect VEGFA immunofluorescence against gRNA1, gRNA2, and gRNA3.

[0079] Fig.10 The figure shows the expression of VEGFA in cells detected by flow cytometry.

[0080] Fig.11 The inhibition effect of VEGFA in HUVECs cells by the CRISPR / Cas9 system containing gRNA2 or gRNA3 was shown.

[0081] Fig.12 Deep sequencing was used to detect the editing effect of the CRISPR / Cas9 system containing gRNA3 on the DNA of VEGFA.

[0082] Fig.13 Shown are the results of ELISA and western blotting experiments of HUVECs after VEGFA inhibition by the CRISPR / Cas9 system containing gRNA3.

[0083] Fig.14 The infection of corneal tissue by the AAV2 / 9-SpCas9 and AAV2 / 9-gRNA3 dual AAV vector systems under confocal microscopy is shown.

[0084] Fig.15 The inhibition of VEGFA protein expression in corneal tissue by the AAV2 / 9-SpCas9 and AAV2 / 9-gRNA3 dual AAV vector systems and the quantitative fluorescence analysis of VEGFA protein are shown under a confocal microscope.

[0085] Fig.16 The inhibition of angiogenesis in a mouse corneal neovascular disease model by the dual AAV vector system of AAV2 / 9-SpCas9 and AAV2 / 9-gRNA3 is shown.

[0086] Fig.17 Quantitative analysis of the inhibitory effects of AAV2 / 9-SpCas9 and AAV2 / 9-gRNA3 dual AAV vector systems on angiogenesis in a mouse corneal neovascular disease model over time is shown.

[0087] Fig.18 The effects of the AAV2 / 9-SpCas9 and AAV2 / 9-gRNA3 dual AAV vector systems on the retinal electrophysiological characteristics of the mouse corneal neovascular disease model are shown.

[0088] Fig.19The effects of the AAV2 / 9-SpCas9 and AAV2 / 9-gRNA3 dual AAV vector system on the corneal pathological structure and pathological structure of important organs in the mouse corneal neovascular disease model are shown. DETAILED DESCRIPTION

[0089] After extensive and in-depth research, the inventors unexpectedly found for the first time the 5'UTR region, exon 1, exon 6, and 3'UTR region of the VEGFA gene (for example, positions 14538-14557, and / or positions 14377-14396, and / or positions 10589-10608, and / or positions 14370-14389, and / or positions 454-473, and / or positions 493-512, and / or positions 1095-1114 of the VEGFA gene (Gene ID: 7422), and the gRNA designed for this position has the characteristics of high editing efficiency and low off-target rate, and the combination of type II Cas protein (such as Cas9) and the gRNA targeting the 5'UTR region, exon 1, exon 6, and 3'UTR region of the VEGF gene of the present invention can effectively prevent and / or treat corneal neovascularization diseases. On this basis, the inventors have completed the present invention.

[0090] Terminology

[0091] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0092] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0093] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0094] Specifically, the gRNA, CRISPR / Cas9 system, and expression vector system provided by the present invention for treating corneal neovascularization diseases can efficiently inhibit the expression of the VEGFA gene in corneal neovascular endothelial cells. When 2.50E+9 vg of AAV2 / 9-CRISPR / Cas9 is subconjunctivally injected into mice with corneal neovascularization disease models once, quantitative detection of corneal neovascularization is performed on the 3rd, 7th, and 10th days after injection. Compared with the control group, the lengths of the neovascularization decreased by 59.74±2.65%, 45.26±0.52%, and 37.71±1.88% respectively. After immunofluorescence staining of corneal neovascularization tissues and scanning with a confocal laser microscope, it was found that compared with the control group, the fluorescence intensity and area of neovascularization in the treated corneal tissues were significantly lower than those of the control group. Image acquisition of the cornea was performed with a slit lamp fibroscope. Compared with the control group, the length and area of corneal neovascularization after treatment were significantly lower than those of the control group.

[0095] Electroretinogram detection was performed on the 4th and 8th weeks after subconjunctival single injection of 2.50E+9 vg of AAV2 / 9-CRISPR / Cas9 into mice with corneal neovascularization disease models. Compared with the control group of mice injected with the same volume of normal saline, no damage to the electroretinogram function of the retina was found due to the treatment. No pathological changes were observed in the corneal tissues, retina tissues, heart, liver, spleen, lungs, and kidneys, and no expression of the recombinant adeno-associated virus containing the system was detected.

[0096] In summary, the present invention provides an efficient and highly safe CRISPR / Cas9 system for treating corneal neovascularization diseases, demonstrating an application prospect in the treatment of corneal neovascularization diseases.

[0097] As used herein, the Chinese name of Pegaptanib is pegaptanib, and its chemical structural formula is:

[0098]

[0099] As used herein, the Chinese name of Aganirsen is aganirsen, and its chemical structural formula is:

[0100]

[0101] Corneal neovascularization diseases

[0102] The cornea is an important refractive structure in the visual system, and its avascular nature is crucial to maintaining corneal transparency. Corneal neovascularization (CNV) has a large number of cases and a high blindness rate. It has always been a hot topic in the field of corneal diseases around the world and is also one of the major eye diseases that urgently need to be solved. Under pathological conditions such as infection, inflammation, hypoxia, trauma, corneal degeneration or tumors, the balance between corneal pro-angiogenic and anti-angiogenic factors is broken, causing vascular endothelial cells to proliferate and invade the corneal stroma, thereby forming CNV. CNV can cause corneal scarring, stromal edema, lipid deposition and keratitis, leading to visual impairment. Many severe cases ultimately require keratoplasty to restore vision. However, CNV is also a risk factor and major complication of corneal transplantation failure. Among the diseased corneas removed by keratoplasty, samples containing CNV account for up to 20%.

[0103] At present, clinical treatments for CNV include corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), photodynamic therapy (PDT), laser photocoagulation, fine needle diathermy, amniotic membrane transplantation, etc. However, these therapies have limited effects, short duration of action (requires multiple administrations), and some serious side effects. For example, corticosteroids can cause high intraocular pressure, cataracts, and superinfection, NSAIDs can cause corneal epithelial damage and delayed epithelial wound healing, laser photocoagulation treatment is prone to recurrence, collagen contraction and damage to the adjacent matrix of the diathermy site, changes in corneal curvature, and even stimulation of further CNV formation. Vascular endothelial growth factor receptor A (VEGFA) is one of the strongest angiogenic factors, and its upregulation is the most important cause of CNV. Inhibitory antibodies targeting the VEGFA signaling pathway (such as aflibercept, ranibizumab, and bevacizumab) have shown good results in the treatment of CNV, but they are only effective for active blood vessels, and the effect is short-lived and incomplete, requiring repeated injections, and CNV may recur. There may also be side effects such as corneal thinning. Therefore, the clinical treatment of CNV is still a huge challenge.

[0104] Gene editing proteins

[0105] In the present invention, the nucleotides of the gene editing protein can be obtained by genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR), etc., and its amino acid sequence can be derived from the nucleotide sequence. The sources of the wild-type gene editing protein include (but are not limited to): Ruminococcus Flavefaciens, Streptococcus pyogenes, Staphylococcus aureus, Acidaminococcus sp, Lachnospiraceae bacterium.

[0106] In a preferred example of the present invention, the gene editing protein includes, but is not limited to, type II Cas protein, preferably Cas9.

[0107] VEGFA gene and its encoded protein

[0108] In the present invention, the terms "VEGFA gene" and "VEGFA polynucleotide" are used interchangeably and refer to nucleic acid sequences having the VEGFA nucleotide sequence.

[0109] The genomic length of the human VEGFA gene is 16,277 bp (Gene ID: 7422) and the location is GRCh38 chr6: 43,770,211-43,786,487.

[0110] The genomic length of the mouse VEGFA gene is 15385 bp (Gene ID: 22339) and the position is GRCm38chr17: 46,016,993..46,032,377.

[0111] Human and mouse VEGFA have a similarity of 44.84% at the DNA level and a protein sequence similarity of 82.53%. It should be understood that when encoding the same amino acid, nucleotide substitutions in codons are acceptable. It should also be understood that when conservative amino acid substitutions are produced by nucleotide substitutions, nucleotide changes are also acceptable.

[0112] In the case of obtaining the amino acid fragment of VEGFA, a nucleic acid sequence encoding it can be constructed based on it, and a specific probe can be designed based on the nucleotide sequence. The full-length nucleotide sequence or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the VEGFA nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art is used as a template to amplify the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the fragments amplified each time are spliced ​​together in the correct order.

[0113] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0114] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0115] At present, the DNA sequence encoding the protein of the present invention (or its fragment, derivative) can be obtained completely by chemical synthesis. Then the DNA sequence can be introduced into various existing DNA molecules (such as vectors) and cells known in the art.

[0116] The polynucleotide sequences of the present invention can be used to express or produce recombinant VEGFA polypeptides by conventional recombinant DNA techniques. Generally, the following steps are involved:

[0117] (1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a human VEGFA polypeptide of the present invention, or a recombinant expression vector containing the polynucleotide;

[0118] (2) Host cells cultured in a suitable culture medium;

[0119] (3) Isolate and purify proteins from culture medium or cells.

[0120] In the present invention, the VEGFA polynucleotide sequence can be inserted into a recombinant expression vector. In short, any plasmid and vector can be used as long as they can replicate and be stable in the host. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0121] Methods well known to those skilled in the art can be used to construct expression vectors containing VEGFA encoding DNA sequences and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site and a transcription terminator for translation initiation.

[0122] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0123] The vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins.

[0124] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells; animal cells, etc.

[0125] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0126] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0127] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0128] Adeno-associated virus

[0129] Because adeno-associated virus (AAV) is smaller than other viral vectors, has no pathogenicity, and can transfect both dividing and non-dividing cells, gene therapy methods based on AAV vectors for genetic diseases have received widespread attention.

[0130] Adeno-associated virus (AAV), also known as adeno-associated virus, belongs to the genus Dependinovirus of the family Parvoviridae. It is the simplest type of single-stranded DNA defective virus discovered so far, and requires a helper virus (usually adenovirus) to participate in replication. It encodes the cap and rep genes in the inverted repeat sequences (ITR) at both ends. ITRs play a decisive role in the replication and packaging of the virus. The cap gene encodes the viral capsid protein, and the rep gene is involved in the replication and integration of the virus. AAV can infect a variety of cells.

[0131] Recombinant adeno-associated virus vector (rAAV) is derived from non-pathogenic wild-type adeno-associated virus. Due to its good safety, wide range of host cells (dividing and non-dividing cells), low immunogenicity, and long-term expression of foreign genes in vivo, it is regarded as one of the most promising gene transfer vectors and has been widely used in gene therapy and vaccine research worldwide. After more than 10 years of research, the biological characteristics of recombinant adeno-associated virus have been deeply understood, especially its application effects in various cells, tissues and in vivo experiments have been accumulated. In medical research, rAAV has been used in the study of gene therapy for a variety of diseases (including in vivo and in vitro experiments); at the same time, as a characteristic gene transfer vector, it is also widely used in gene function research, construction of disease models, preparation of gene knockout mice, etc.

[0132] In a preferred embodiment of the present invention, the vector is a recombinant AAV vector. AAV is a relatively small DNA virus that can be integrated into the genome of the cells they infect in a stable and site-specific manner. They can infect a large series of cells without any effect on cell growth, morphology or differentiation, and they do not seem to be involved in human pathology. The AAV genome has been cloned, sequenced and characterized. AAV contains an inverted terminal repeat (ITR) region of about 145 bases at each end, which serves as the origin of viral replication. The rest of the genome is divided into two important regions with encapsidation function: the left part of the genome containing the rep gene involved in viral replication and viral gene expression; and the right part of the genome containing the cap gene encoding the viral capsid protein.

[0133] AAV vectors can be prepared using standard methods in the art. Any serotype of adeno-associated virus is suitable. Methods for purifying vectors can be found in, for example, U.S. Patent Nos. 6,566,118, 6,989,264, and 6,995,006, the disclosures of which are incorporated herein by reference in their entirety. The preparation of hybrid vectors is described, for example, in PCT Application No. PCT / US2005 / 027091, the disclosures of which are incorporated herein by reference in their entirety. The use of vectors derived from AAV for transporting genes in vitro and in vivo has been described (see, for example, International Patent Application Publication Nos. WO91 / 18088 and WO93 / 09239; U.S. Patent Nos. 4,797,368, 6,596,535, and 5,139,941, and European Patent No. 0488528, all of which are incorporated herein by reference in their entirety). These patent publications describe various AAV-derived constructs in which the rep and / or cap genes are deleted and replaced by the genes of interest, and the use of these constructs to transport the genes of interest in vitro (into cultured cells) or in vivo (directly into an organism). Replication-deficient recombinant AAV can be prepared by co-transfecting the following plasmids into a cell line infected with a human helper virus (e.g., adenovirus): a plasmid containing a nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes (rep and cap genes). The resulting AAV recombinants are then purified by standard techniques.

[0134] In some embodiments, the recombinant vector is encapsidated into a viral particle (e.g., AAV virus particles including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16). Thus, the present disclosure includes recombinant viral particles (recombinant because they contain a recombinant polynucleotide) containing any of the vectors described herein. Methods for producing such particles are known in the art and are described in U.S. Pat. No. 6,596,535.

[0135] Pharmaceutical composition

[0136] By using the protein of the present invention and various conventional screening methods, substances that interact with the VEGFA gene or protein, especially inhibitors, etc., can be screened out.

[0137] The VEGFA inhibitors (or antagonists) that can be used in the present invention include any substances that can inhibit the expression and / or activity of the VEGFA gene or its encoded protein.

[0138] For example, the VEGFA inhibitor includes an antibody to VEGFA, an antisense RNA to VEGFA nucleic acid, siRNA, shRNA, miRNA, a gene editor, or an activity inhibitor of VEGFA. A preferred VEGFA inhibitor refers to a gene editor (e.g., a gene editor comprising a gene editing protein and a gRNA) that can inhibit VEGFA expression.

[0139] Preferably, the VEGFA inhibitors of the present invention include inhibitors targeting the VEGFA gene sequence ((Gene ID: 7422) positions 14538-14557, and / or positions 14377-14396, and / or positions 10589-10608, and / or positions 14370-14389, and / or positions 454-473, and / or positions 493-512, and / or positions 1095-1114). In a preferred embodiment, the method and steps for inhibiting VEGFA include neutralizing VEGFA protein with antibodies to VEGFA, and silencing the VEGFA gene with shRNA or siRNA or gene editors carried by viruses (such as adeno-associated viruses).

[0140] The inhibition rate of VEGFA is generally at least 50%, preferably 60%, 70%, 80%, 90%, 95%. The inhibition rate of VEGFA can be controlled and detected based on conventional techniques, such as flow cytometry, fluorescence quantitative PCR or Western blot.

[0141] The inhibitors of the VEGFA protein of the present invention (including antibodies, antisense nucleic acids, gene editors and other inhibitors), when applied (administered) for treatment, can inhibit the expression and / or activity of the VEGFA protein, thereby preventing and / or treating corneal neovascularization diseases. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary with the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): topical, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, topical administration, autologous cell extraction and culture followed by reinfusion, etc.

[0142] The present invention also provides a pharmaceutical composition, which contains a safe and effective amount of an inhibitor of the present invention (such as an antibody, a gene editor, an antisense sequence (such as siRNA), or an inhibitor) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram to 10 mg per kilogram of body weight per day.

[0143] The main advantages of the present invention include:

[0144] (1) The present invention unexpectedly found for the first time the 5'UTR region, exon 1, exon 6, and 3'UTR region of the VEGF gene (for example, positions 14538-14557, and / or positions 14377-14396, and / or positions 10589-10608, and / or positions 14370-14389, and / or positions 454-473, and / or positions 493-512, and / or positions 1095-1114 of the VEGF gene (Gene ID: 7422), and the gRNA designed for this position has the characteristics of high editing efficiency and low off-target rate, and the type II Cas protein (such as Cas9) and the 5'UTR region, exon 1, exon 6, and 3'UTR region of the VEGF gene targeted by the present invention (for example, positions 14538-14557, and / or positions 14377-14396, and / or positions 10589-10608, and / or positions 14370-14389, and / or positions 454-473, and / or positions 493-512, and / or positions 1095-1114 of the VEGF gene (Gene ID: 7422) are combined. The gRNA combination of positions 14538-14557, and / or positions 14377-14396, and / or positions 10589-10608, and / or positions 14370-14389, and / or positions 454-473, and / or positions 493-512, and / or positions 1095-1114 of IBD PGC-MS / MDA (ID: 7422) can effectively prevent and / or treat corneal neovascularization diseases.

[0145] (2) The present invention provides a gRNA that can effectively inhibit the expression of the VEGFA gene, and can deliver the CRISPR / Cas9 system and gRNA that play a gene editing role into the animal body through a dual AAV expression vector system to play a role in editing the VEGFA gene, and provides the use of the system for preparing gene therapy drugs for treating corneal neovascularization diseases.

[0146] (3) The present invention provides an efficient and highly safe CRISPR / Cas9 system for treating corneal neovascularization diseases, demonstrating its application prospects in the treatment of corneal neovascularization diseases.

[0147] (4) The present invention provides a highly efficient and safe gene editing system that can not only inhibit the expression of the VEGFA gene in a mouse model of corneal neovascular disease, but also simultaneously target the VEGFA gene in the human genome, thereby effectively solving the problem of the transition of gene editing drug development from animal experiments to clinical transformation.

[0148] The present invention will be further described below in conjunction with specific implementation. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed 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 according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0149] Unless otherwise specified, the reagents and materials used in the examples of the present invention are all commercially available products.

[0150] Example 1 Construction of CRISPR / Cas9 system targeting VEGFA gene

[0151] 1. Target selection of VEGFA gene by CRISPR / Cas9 system

[0152] The 5'UTR, exon region and 3'UTR region of the VEGFA gene (Gene ID: 7422) were selected as targets, and gene sites with "NGG" protospacer adjacent motif (PAM) were searched. Seven target sites with high editing efficiency and low off-target rate that simultaneously targeted the common conservative sequences of human VEGFA gene and mouse VEGFA gene were selected. The specific positions corresponding to gRNA are shown in Table A. Cas-OFFinder was used to predict possible off-target sites. The VEGFA gene site information where the gRNA target site is located is shown in Figure 1 .

[0153] Table A

[0154]

[0155] 2. Construction of CRISPR / Cas9 system expression vector

[0156] Primers were synthesized according to the DNA sequence information of gRNA, and the synthesized fragments were annealed into double-stranded DNA by PCR instrument, and cloned into CRISPR / Cas9 backbone plasmid (purchased from addgene, https: / / www.addgene.org / ) by directional cloning technology. The CMV promoter was used to drive the expression of Cas9. The green fluorescent protein gene EGFP was used to fluorescently label Cas9 protein for tracing to obtain the expression abundance and spatiotemporal localization information of Cas9. The constructed plasmid was identified by Sanger sequencing. The sequence information of each site is shown in Table 1.

[0157] Table 1 gRNA sequences designed for VEGFA gene

[0158]

[0159]

[0160] The gRNA double-stranded in vitro synthesis reaction system is 5μl 2*quick ligase buffer, 1μl 100μM gRNA positive single strand, 1μl 100μM gRNA antisense single strand, 0.5μl T4 PNK, 2.5μl ddH2O. The reaction program is set in a PCR instrument, 37℃ reaction for 30 minutes, 95℃ reaction for 5 minutes, and moved to room temperature for 2 hours.

[0161] The px458-spCas9 plasmid (purchased from addgene, https: / / www.addgene.org / ) was digested with BbsI ( Figure 2 ) as the backbone of the gRNA expression vector. The reaction system is 0.5ul BbsI, 2ug px458-spCas9 plasmid, 4ul 10*NEB Cutsmart buffer, and ddH2O to make up the reaction system to 50ul. The reaction procedure is to react in a PCR instrument at 37°C for 2 hours.

[0162] DNA recovery of enzyme digestion products

[0163] The specific steps are as follows: Use a clean blade to cut the target band from the agarose gel electrophoresis gel and place it in a 2.0 ml EP tube; add 300 μl of sol-binding solution for every 100 mg of gel and heat it in a 56°C water bath for 10 min; place the adsorption column in the collection tube, and after the gel is completely dissolved, add the solution to the adsorption column, let it stand at room temperature for 1 min, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid in the collection tube; add 500 μl of rinsing solution, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid in the collection tube; add 500 μl of rinsing solution again, Centrifuge at 12000rpm for 30sec, discard the waste liquid in the collection tube; put the adsorption column back into the collection tube, centrifuge at 12000rpm for 2min to remove the rinse solution; put the adsorption column into a 1.5ml EP tube, add 30ul elution solution in the middle of the adsorption membrane, let it stand at room temperature for 2min, and centrifuge at 12000rpm for 1min; aspirate the resulting solution from the EP tube, drop it again in the middle of the original adsorption membrane, let it stand at room temperature for 1min, and centrifuge at 12000rpm for 1min; use NanoDrop to measure the DNA concentration in the resulting solution.

[0164] Connect gRNA and vector backbone

[0165] The digested product after gel recovery and the primer annealing product diluted 100 times were connected using T4 DNA ligase to obtain the expression vector of sgRNA. The reaction system was 1ul gRNA, 0.5ul T4 DNA ligase, 50ng px458-spCas9 backbone fragment, 1ul 10*T4 DNA ligase buffer, and finally ddH2O was used to fill the system to 10ul. The reaction procedure was in a PCR instrument, 22℃ for 30min.

[0166] 3. Transform the ligation product into competent cells

[0167] The specific implementation steps are as follows: 2ul of the ligation product was transformed into 25ul of DH5a competent cells (purchased from Beijing Qingke Biotechnology), and then placed on ice for 30min. Then, the EP tube was placed in a 42℃ water bath for 90 seconds without shaking. After heat shock, it was immediately placed on ice and allowed to stand for 3min. 75ul of LB medium with ampicillin antibiotics was added, and the plate was directly coated after mixing, and placed in the incubator overnight. Then, 5 single clones were selected and placed in a sterile shaking tube containing 12ml of LB medium with 4ml of ampicillin antibiotics, and the tube was shaken at 220 rpm for 12 hours, and 1ml of the bacterial solution was sent for sequencing and identification.

[0168] Example 2 Detection of the efficiency of CRISPR / Cas9 system in inhibiting VEGFA gene expression in human kidney embryo cells 1. Cell culture

[0169] Human kidney embryonic cells (HEK-293T) (purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee) were cultured in high-glucose DMEM medium (purchased from Gibco Thermo Fisher Scientific) supplemented with 10% fetal bovine serum. All cells were cultured at 37° C. in a humidified environment of 5% CO 2 .

[0170] 2. Cell transfection with CRISPR / Cas9 system

[0171] Cells were cultured in 24-well plates. Lipofectamine 3000 (purchased from Thermo Fisher Scientific) and CRISPR / Cas9 system plasmid (Lipofectamine 3000 2μl / well, plasmid 1μg / well) were thoroughly mixed in Opti-MEM (purchased from Thermo Fisher Scientific), while the control group only added Opti-MEM and Lipofectamine3000. After incubation for 6 hours, fresh culture medium was replaced. Two days later, cells were photographed under an immunofluorescence microscope to determine the transfection efficiency.

[0172] 3. Detection of VEGFA gene expression inhibition efficiency

[0173] T7EI can identify genomic DNA sites edited by the CRISPR / Cas9 system and cut sites with traces of editing. If the CRISPR / Cas9 system can edit the VEGFA target site with high efficiency, in the T7EI experiment, there will be extra bands below the uncut bands ( Figure 3 In the Elisa experiment, the expression level of VEGFA in the cell supernatant decreased ( Figure 4 ), among which gNRA2 and gRNA3 had statistically significant effects on the inhibition of VEGFA expression. gRNA1, gRNA2, and gRNA3 were selected to lyse cells, separate and collect genomic DNA for PCR and then sent to Sanger sequencing. The results showed that a hybrid peak appeared upstream of the editing site ( Figure 5 For Western blot analysis, cells were homogenized in a buffer solution (10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 50 mM NaCl, 50 mM NaF, 1% Triton x-100,

[0174] 20μg / ml aprotinin, 2mM Na3VO4, and 1mM phenylmethylsulfonyl fluoride) from 5× protein sample buffer (25mM EDTA (pH=7.0), 10% sodium dodecyl sulfate (SDS), 500mM dithiothreitol, 50% sucrose, 500mM Tris-HCl (pH=6.8), and 0.5% bromophenol blue). The lysate was boiled for 5 minutes and centrifuged at 12000g for 10 minutes. The proteins in the samples were separated by 10% SDS-PAGE, transferred to polyvinylidene difluoride membrane, and analyzed by western blot. The experiment was repeated at least three times. The signal intensity was determined by density using Image J software. The results show that the CRISPR / Cas9 system achieved efficient VEGFA target gene editing compared with the control group, and VEGFA expression was strongly inhibited ( Figure 6 ), quantitative analysis of protein immunoblotting showed that compared with the control group, all three gRNAs achieved a relatively strong inhibition of VEGFA expression ( Figure 7 ).

[0175] 4. Off-target Detection

[0176] Cas-OFFinder was used to predict possible off-target sites, and T7EI detection was performed on the predicted target sites. No editing was found at the potential off-target sites. Figure 8 ).

[0177] Example 3 Detection of the efficiency of CRISPR / Cas9 system in inhibiting VEGFA gene expression in vascular endothelial cells

[0178] 1. Cell Culture

[0179] Primary human umbilical vein endothelial cells (HUVECs) (purchased from Thermo Fisher Scientific) were cultured in endothelial culture medium supplemented with 10% fetal bovine serum (FBS) and endothelial growth factor. All cells were cultured at 37°C in a humidified environment with 5% CO2.

[0180] 2. Cell transfection with CRISPR / Cas9 system

[0181] Cells were cultured in 24-well plates. Lipofectamine 3000 and the CRISPR / Cas9 system plasmid containing gRNA2 or gRNA3 screened in Example 1 (Lipofectamine 3000 2 μl / well, plasmid 1 μg / well) were fully mixed in Opti-MEM, while the control group only added Opti-MEM and Lipofectamine 3000. After incubation for 6 hours, fresh culture medium was replaced. Two days later, cells were photographed under an immunofluorescence microscope to determine the transfection efficiency.

[0182] 3. Detection of VEGFA gene expression inhibition efficiency

[0183] Immunofluorescence staining of intracellular VEGFA expression was performed and scanning was performed using a laser confocal microscope. The results showed that compared with the control group, the CRISPR / Cas9 system plasmid containing gRNA2 or gRNA3 showed a significant inhibitory effect on the expression of intracellular VEGFA, and the inhibitory effect of the CRISPR / Cas9 system plasmid containing gRNA3 was better than that of the CRISPR / Cas9 system plasmid containing gRNA2 ( Fig. 9 Flow cytometry showed that VEG FA was significantly reduced in HUVECs transduced with CRISPR / Cas9 system plasmids containing gRNA2 or gRNA3 ( Fig.10 In addition, ELISA and immunofluorescence results also showed that HUVECs transduced with the CRISPR / Cas9 system plasmid containing gRNA3 had less VEGFA content than those transduced with the control plasmid ( Fig.11 ). Deep sequencing data showed that more than 70% of VEGFA DNA was cut by SpCas9 / sgRNA3 ( Fig.12 ), the above data indicate that the CRISPR / Cas9 system plasmid containing gRNA3 can better inhibit the expression of VEGFA protein than the CRISPR / Cas9 system containing gRNA2.

[0184] Example 4 CRISPR / Cas9 system containing gRNA3 can effectively inhibit VEGFA gene expression in mouse cells

[0185] We tested whether the CRISPR / Cas9 system containing gRNA3 could work in mouse cells. The VEGFA content in the supernatant and cell lysate of RAW264.7 cells transfected with the CRISPR / Cas9 system containing gRNA3 was significantly reduced, indicating that the CRISPR / Cas9 system containing gRNA3 can effectively edit VEGFA in mice ( Fig.13 , ad).

[0186] Example 5 Dual AAV vector delivery system containing CRISPR / Cas9 system of gRNA3 can edit VEGFA gene and prevent suture-induced corneal neovascularization

[0187] Establishment and medication of suture-induced corneal neovascularization (CNV): A suture-induced corneal neovascularization model was established to evaluate the efficacy of the AAV-CRISPR / SpCas9-VEGFA system in vivo. After general anesthesia with isoflurane and local anesthesia with propacaine, mice were placed in a lateral recumbent position. A circular filter paper with a diameter of 2 mm was pasted to the center of the cornea, and the edge of the filter paper was used to determine the position of the superior temporal needle insertion. Only the right eye of each mouse was sutured. All mice were randomly divided into groups and injected with a total dose of 5 μl of solution under the conjunctiva: (1) PBS, (2) AAV-control, or (3) AAV2 / 9 dual vector system containing the CRISPR / Cas9 system of gRNA3. The CNV was observed under a slit lamp biomicroscope on days 3, 7, and 10 to evaluate the anti-CNV effect. The length and area of ​​the CNV were determined using Image Pro Plus6 and calculated according to the previous sector area method. The formula is as follows: S = C ÷ 12 × π × [r 2 -(rL) 2 ], S is the CNV area, C is the sector CNV clock, r is the corneal radius, and L is the average length of the new blood vessels at the beginning of the corneal limbus. The dual AAV vector system of AAV2 / 9-SpCas9 and AAV2 / 9-gRNA3 targeting mouse VEGFA (1:1, 2.5E+9vg) was injected into the subconjunctiva of the mouse close to the corneal limbus. 28 days after the injection, sutures were surgically placed to induce corneal neovascularization. The results showed that EGFP was expressed in the cornea after injection ( Fig.14 , ac), illustrating that AAV2 / 9-spCas9 / AAV2 / 9-VEGFA dual AAV vector transduces corneal tissue.

[0188] In addition, immunofluorescence and ELISA analysis also showed that VEGFA in corneal tissue was reduced after injection of the VEGFA-targeting AAV2 / 9-SpCas9 and AAV2 / 9-gRNA3 dual AAV vector system compared with the control group ( Fig.15 ).

[0189] Seven days after suturing, corneal neovascularization was observed using a slit lamp. Fig.16 As shown in the white dashed box in the middle, injection of control AAV did not affect corneal neovascularization, while injection of the dual AAV vector system of AAV2 / 9-SpCas9 and AAV2 / 9-gRNA3 targeting VEGFA inhibited suture-induced neovascularization ( Fig.16 ). There was less corneal neovascularization at 3, 7, and 10 days after injection of double AAV suture targeting VEGFA ( Fig.17 ). These results indicate that the dual AAV vector system of AAV2 / 9-SpCas9 and AAV2 / 9-gRNA3 targeting VEGFA can effectively inhibit angiogenesis.

[0190] Example 6 Subconjunctival injection of AAV2 / 9-SpCas9 and AAV2 / 9-gRNA3 dual AA V vector system targeting VEGFA does not interfere with retinal function and affect other important organs

[0191] We investigated whether inhibition of VEGFA expression in ocular tissues using a dual AAV vector system of AAV2 / 9-SpCas9 and AAV2 / 9-gRNA3 targeting VEGFA would affect retinal function. We examined retinal function using electroretinography (ERG) and retinal structure using H&E staining. The ocular safety of the AAV-CRISPR / SpCas9-VEGFA system via subconjunctival injection was evaluated by electroretinography (ERG), rti-port, and histopathological observation. Healthy male mice were selected and anesthetized systemically and locally. 5 μL of the AAV-CRISPR / SpCas9-VEGFA system was injected subconjunctivally in the temporal quadrant of the right eye of the mice, and the same amount of PBS was injected into the left eye as a control. The RETI visual function assessment system was used at 4 and 8 weeks after injection. After the mice were dark-adapted overnight, 5% (w / v) chloral hydrate (0.08 mL / 10 g) was injected intraperitoneally for anesthesia.

[0192] The pupil was then dilated with tropicamide, and the local ocular surface was anesthetized with propacaine and moistened with 0.1% sodium hyaluronate (Santen, Japan) for better electrical conduction. Subsequently, a ground needle electrode was inserted under the skin of the tail, two reference needle electrodes were placed under the skin of the cheeks on both sides, and two gold wire ring electrodes were carefully placed on the surface of both eyes. The full-field electrogram was recorded according to the standard protocol of the International Society for Clinical Electrophysiology of Vision (ISCEV). The retinal ERG a wave and b wave were recorded and averaged. The results showed that there was no significant difference in the ERG a and b waves at 4 or 8 weeks in the eyes injected with the dual AAV vector system of AAV2 / 9-SpCas9 and AAV2 / 9-gRNA3 targeting VEGFA compared with the control group ( Fig.18 ).

[0193] We next evaluated whether the dual AAV would affect other organs after subconjunctival injection. Histopathological observations were performed 12 weeks after injection. The mouse eyes and organs were obtained for paraffin pathological staining. 5 μm sections were taken and stained with hematoxylin-eosin to observe the structural morphology of eye and organ tissues. The results showed that compared with the control group, there were no significant changes in these examined tissues after subconjunctival injection of the AAV2 / 9-SpCas9 and AAV2 / 9-gRNA3 dual AAV vector system targeting VEGFA ( Fig.19 ). All these data together showed that no toxicity was detected after subconjunctival injection of the dual AAV vector system of AAV2 / 9-SpCas9 and AAV2 / 9-gRNA3 targeting VEGFA.

[0194] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A composition, characterized in that include: (a) a gene editing protein or an expression vector thereof, wherein the gene editing protein comprises a type II Cas protein; and (b) gRNA or its expression vector, wherein the gRNA guides the gene editing protein to specifically bind to the DNA of the VEGFA gene, and the region targeted by the gRNA is the 5'UTR region, exon 1, exon 6, and 3'UTR region of the VEGFA gene sequence.

2. The composition according to claim 1, characterized in that The region targeted by the gRNA is positions 14538-14557, and / or positions 14377-14396, and / or positions 10589-10608, and / or positions 14370-14389, and / or positions 454-473, and / or positions 493-512, and / or positions 1095-1114 of the VEGFA gene sequence (Gene ID: 7422).

3. The composition according to claim 1, characterized in that The gene editing protein includes Cas9.

4. The composition according to claim 3, characterized in that The source of the Cas9 is selected from the following group: Ruminococcus Flavefaciens, Streptococcus pyogenes, Staphylococcus aureus, Acidaminococcus sp, Lachnospiraceae bacterium, or a combination thereof.

5. The composition according to claim 1, characterized in that The nucleotide sequence of the gRNA is selected from the group consisting of SEQ ID NO.: 1, 2, 3, 4, 5, 6 and 7.

6. The composition according to claim 1, characterized in that The composition further comprises: (c) Other drugs for the prevention and / or treatment of corneal neovascularization diseases.

7. The composition according to claim 1, characterized in that The expression vector includes a viral vector.

8. A medicine box, characterized in that: include: (a1) a first container, and a gene editing protein or its expression vector, or a drug containing the gene editing protein or its expression vector, located in the first container, wherein the gene editing protein comprises a type II Cas protein; (b1) a second container, and a gRNA or its expression vector located in the second container, or a drug containing the gRNA or its expression vector, wherein the gRNA guides the gene editing protein to specifically bind to the DNA of the VEGF A gene, and the region targeted by the gRNA is the 5'UTR region, exon 1, exon 6, and 3'UTR region of the VEGFA gene sequence.

9. Use of the composition according to claim 1 or the kit according to claim 8, characterized in that: Used for preparing drugs for preventing and / or treating corneal neovascularization diseases.

10. An AAV vector, characterized in that include: (a) a coding sequence of a gene editing protein, wherein the gene editing protein comprises a type II Cas protein; and (b) gRNA, wherein the gRNA guides the gene editing protein to specifically bind to the DNA of the VEGFA gene, and the region targeted by the gRNA is the 5'UTR region, exon 1, exon 6, and 3'UTR region of the VEGFA gene sequence.

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