Application of growth hormone receptor antagonist and nucleic acid molecule in preparation of medicine for preventing or treating retinal neovascularization diseases

Abstract

The invention discloses application of a growth hormone receptor antagonist and a nucleic acid molecule in preparation of a medicine for preventing or treating retinal neovascularization diseases, and relates to the technical field of retinal neovascularization. The invention finds that the growth hormone receptor antagonist or the blocking reagent can effectively intervene the occurrence and development of the neovascular retina disease and improve the symptom of the disease. In addition, the transcription level of the encoding gene of the growth hormone receptor is reduced in a gene editing mode, and the pathological vascular change of the retinal neovascular disease can be effectively improved by knocking out the retina GHR, so that the pathological neovascular growth of the retina is reduced, and vascular exudation and inflammatory response are reduced. The invention provides a new direction for the treatment of retinal neovascular diseases, and provides a new choice for patients intolerant or unresponsive to anti-VEGF (Vascular Endothelial Growth Factor) treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of retinal neovascularization, and in particular to the use of a growth hormone receptor antagonist and a nucleic acid molecule in the preparation of a drug for preventing or treating retinal neovascularization diseases. Background Art

[0002] Retinal neovascularization is a major pathological change in retinal diseases such as neonatal retinopathy, diabetic retinopathy, retinal vein occlusion, and age-related macular degeneration. It often leads to secondary problems such as retinal exudation, hemorrhage, and retinal detachment, resulting in damage to retinal structure and function, and subsequently causing visual impairment and even blindness. Neovascularization is usually a compensatory or adaptive response of tissues to stimuli such as hypoxia, ischemia, and inflammation. It is a chronic change that is difficult to recover naturally. It is mainly manifested by excessive proliferation, differentiation, and migration of vascular endothelial cells, and a loss of the number and function of vascular wall cells, which ultimately leads to abnormal arteriovenous fusion, clustered growth, increased permeability, and the formation of a new vascular network.

[0003] Currently, the main treatments for retinal neovascularization are medication and surgery. Medication primarily targets VEGF, a factor that regulates retinal neovascularization. VEGF or its receptor inhibitors are used to inhibit VEGF activity, thereby controlling neovascularization. However, only a subset of patients with this disease respond to anti-VEGF drugs, and treatment outcomes vary widely. These drugs can lead to complications such as the need for repeated treatments, retinal atrophy, and cardiovascular disease. Intraocular injections of steroids such as triamcinolone acetonide can also be used to reduce intraocular vascular inflammation. These drugs are primarily used to treat macular edema that is intolerant or unresponsive to anti-VEGF therapy, but their use is relatively limited. Surgical treatment is indicated for patients with more severe complications of retinal neovascularization, such as vitreous hemorrhage, proliferative membrane formation, and retinal detachment. Surgical options include retinal laser photocoagulation and vitrectomy. These procedures carry the risk of surgically induced retinal damage. For patients without surgical indications, the benefits of treatment are low.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide the use of growth hormone receptor antagonists and nucleic acid molecules in the preparation of drugs for preventing or treating retinal neovascular diseases, so as to effectively intervene in the occurrence and development of neovascular retinal diseases, improve disease symptoms, and expand clinical treatment ideas for retinal neovascular diseases.

[0006] The present invention is achieved in that: In a first aspect, the present invention provides a use of a growth hormone receptor antagonist in the preparation of a medicament for preventing or treating retinal neovascularization diseases.

[0007] In a second aspect, the present invention provides the use of a nucleic acid molecule, a recombinant vector or a gene editing system targeting a growth hormone receptor in the preparation of a drug for preventing or treating retinal neovascularization. The nucleic acid molecule targets the coding gene of the growth hormone receptor to perform gene knockout, site-directed base change, or site-directed insertion on the coding gene of the growth hormone receptor to achieve downregulation of the transcription level of the coding gene of the growth hormone receptor; the recombinant vector includes a nucleic acid molecule, and the gene editing system includes: a gene editing protein and a nucleic acid molecule, and the gene editing protein is selected from a Cas protein or a fusion of a Cas protein and a cytidine or adenosine deaminase.

[0008] In a third aspect, the present invention provides the use of a VEGF antagonist and a growth hormone receptor antagonist or the above-mentioned growth hormone receptor-targeting nucleic acid molecule, recombinant vector or gene editing system in the preparation of a drug for the combined treatment of retinal neovascular diseases.

[0009] The present invention has the following beneficial effects: This study, focusing on the growth hormone (GH) receptor, uncovered the crucial role of the growth hormone receptor (GHR) in retinal neovascular diseases through multiple approaches, including gene knockout and receptor antagonists. This discovery led to the discovery that GH receptor antagonists or blocking agents can effectively intervene in the development and progression of neovascular retinal diseases, ameliorate disease symptoms, and expand the clinical treatment strategy for retinal neovascular diseases. Furthermore, knocking out retinal GHR through gene editing, which reduces transcription levels of the GH receptor-encoding gene, has also been shown to effectively ameliorate the pathological vascular changes in retinal neovascular diseases, reduce the growth of pathological retinal neovascularization, and alleviate vascular exudation and inflammatory responses. This study provides a new approach for the treatment of retinal neovascular diseases and offers a new option for patients who are intolerant of or unresponsive to anti-VEGF therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0011] Figure 1 For GHR creERT2 ;Infographic diagram of mT / mG mouse construction (A), diagram of GHR expression in the retina (B), and diagram of changes in GHR expression in the retina at different developmental time points (C); Figure 2 In the OIR model, conditional knockout mice with PDGFR-β creERT2 ; GHR (fl / fl) and GHR creERT2 ; mT / mG;DTA gene mice were induced at P12-P15, and the vascular morphology of the retina after GHR knockout; Figure 3 In the OIR model, conditional knockout mice with PDGFR-β creERT2 ; Comparison of vascular exudative index (Fibrinogen) test results of GHR (fl / fl) mice; Figure 4 Construct information for GHR (fl / fl) and DTA transgenic mice; Figure 5 PDGFR-β creERT2 ;GHR(fl / fl) mice and GHR creERT2 mT / mG; After GHR was knocked out during retinal development in DTA mice, the results of GHR expression changes and the results of deep (DL) and superficial (SL) vascular network area changes at P10 were shown; Figure 6 Build an infographic for the OIR model; Figure 7 The figure shows the expression results of GHR in the retina of OIR model mice; Figure 8 This figure shows the changes in retinal neovascularization after the use of two GHR blockers, ATL1103 and GH-G120R, in the OIR model; Figure 9 Figure 3 shows the changes in retinal neovascularization after systemic or intraocular rhGH injection in the OIR model at P14. DETAILED DESCRIPTION

[0012] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided to illustrate, not to limit, the present invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0013] As used herein, the term "growth hormone" refers to a peptide hormone secreted by the pituitary gland that, in addition to stimulating body growth, also has metabolic regulation functions. Growth hormone may be specifically human growth hormone (hGH), and it is well known that hGH consists of 191 amino acids.

[0014] As used herein, the term "growth hormone mutant" refers to a growth hormone in which one or more amino acids in the amino acid sequence are substituted by another amino acid. In other words, it refers to a growth hormone having one or more amino acid substitutions.

[0015] Specifically, the substitution may include substitution of the 120th amino acid in the amino acid sequence of growth hormone (more specifically, substitution with lysine or arginine). In addition, the substitution may include substitution of the 46th amino acid (more specifically, substitution with lysine).

[0016] In this specification, the term "Cas9 protein" refers to the main protein component of the CRISPR / Cas9 system, which forms a complex with crRNA and tracrRNA to form an activated endonuclease or nickase. Cas9 protein or gene information can be obtained from known databases such as the GenBank of the National Center for Biotechnology Information (NCBI), but any substance that can have target-specific nuclease activity together with the guide RNA can be included in the scope of the present invention. In addition, the Cas9 protein can be connected to a protein transduction domain. The protein transduction domain can be a trans-transcription activator (Trans-Activator of Transcription: TAT) protein derived from polyarginine or human immunodeficiency virus (Human immunodeficiency virus: HIV), but is not limited thereto. In addition, those skilled in the art can appropriately connect additional domains to the Cas9 protein according to the purpose.

[0017] Cas9 protein can include wild-type Cas9, inactivated Cas9 (dCas9), all variants of Cas9, such as Cas9 nickase. The inactivated Cas9 can be a FokI nuclease (RNA-guidedFokINuclease: RFN) in which the FokI nuclease domain is connected to the RNA guide of dCas9 or one in which a transcription activator or repressor domain is connected to dCas9, and the Cas9 nickase can be D10ACas9 or H840ACas9, but is not particularly limited thereto. Specifically, the Cas9 can be any one or more selected from the group consisting of SpCas9, VRQR variants, SpCas9-NG, SpCas9-NRRH, SpCas9-NRTH, SpCas9-NRCH, SpG, SpRY and Sc++.

[0018] The source of the Cas protein is also not limited. For example, the Cas9 protein can be derived from Streptococcus pyogenes, Francisella novicida, Streptococcus thermophilus, Legionella pneumophila, Listeria innocua, or Streptococcus mutans.

[0019] Non-limiting examples of Cas proteins include: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified forms thereof. These enzymes are known; for example, the amino acid sequence of the Streptococcus pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2. In some embodiments, an unmodified CRISPR enzyme, such as Cas9, has DNA cleavage activity. In some embodiments, the CRISPR enzyme is Cas9, and can be Cas9 from Streptococcus pyogenes or Streptococcus pneumoniae. The Cas protein can also be selected from Cas12a or Cas13a.

[0020] The term "treating" a disease or condition means eliminating, inhibiting, alleviating or relieving the disease or condition, and the term "preventing" means avoiding and preventing the disease or condition or causing the disease or condition not to occur or appear.

[0021] In a first aspect, the present invention provides a use of a growth hormone receptor antagonist in the preparation of a medicament for preventing or treating retinal neovascularization diseases.

[0022] The inventors have discovered that growth hormone receptor antagonists can effectively intervene in the occurrence and development of neovascular retinal diseases, effectively improve the pathological vascular changes of retinal neovascular diseases, and have good application prospects in the prevention or treatment of retinal neovascularization.

[0023] In a preferred embodiment of the present invention, the growth hormone receptor antagonist is selected from antisense oligonucleotides or GH analogs targeting growth hormone receptor mRNA.

[0024] The antisense oligonucleotide is ATL1103, and the GH analog is selected from a growth hormone mutant or pegvisomant.

[0025] The growth hormone mutant is selected from at least one of the following: H18D, H21N, Q46K, F54P, R64K, G120K or G120R, R167N, K168A, D171S, K17R, E174S, F176Y and I179T, and the growth hormone mutant is a mutation based on the growth hormone amino acid sequence; the growth hormone amino acid sequence is shown in SEQ ID NO.1.

[0026] SEQ ID NO.1: MATGSRTSLLLAFGLLCLPWLQEGSAFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLI QSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF.

[0027] ATL1103 (oligonucleotide drug) and GH-G120R (GH analog or pegvisomant). ATL1103 binds to growth hormone receptor (GHR) mRNA, activating ribonuclease H, cleaving the complex and degrading GHR mRNA. This process blocks GHR gene transcription and inhibits GHR synthesis. GH-G120R is a GH hormone analogue derived by replacing the glycine residue at position 120 with a lysine residue and an arginine residue, respectively. This analogue binds tightly to the receptor at site 1 while inhibiting binding at site 2. While the analogue can bind to the receptor, it cannot activate normal conformational changes in the receptor, resulting in reduced growth activity and an antagonistic effect against rhGH.

[0028] G120R has a significant antagonistic effect on the growth hormone receptor (GHR). Blocking or inhibiting GHR not only reduces pathological blood vessels but also reduces vascular exudation and inflammatory responses. Patent CN201880083338.4 discloses that mutations at these positions can enhance binding to the growth hormone receptor and exhibit long-lasting antagonism. Therefore, at least one of these GH analogs is expected to have promising applications in treating retinal neovascularization.

[0029] Pegvisomant and GH-G120R are both GH analogues, and the effect of pegvisomant is more lasting.

[0030] In a preferred embodiment of the present invention, retinal neovascular diseases include but are not limited to neonatal retinopathy, diabetic retinopathy, retinal vein occlusion or age-related macular degeneration.

[0031] In a preferred embodiment of the present invention, the application includes at least one of the following methods: (1) Reduce the level of GHR in the subjects' retinal pericytes; (2) reduce the level of GHR-positive cells in the subjects' retina; (3) Slowing down the development of retinal blood vessels; (4) Reduce the number of deep retinal capillaries; (5) Reduce retinal pathological neovascularization; (6) Inhibit retinal vascular leakage and inflammation; (7) Reduce the area of retinal neovascularization; (8) Treatment of patients with retinal neovascularization who are intolerant or unresponsive to VEGF antagonists.

[0032] The use of growth hormone receptor antagonists can alleviate, improve or treat retinal neovascularization diseases in subjects, which is within the scope of protection of the present invention.

[0033] In a preferred embodiment of the present invention, the drug further comprises pharmaceutically acceptable excipients, including but not limited to conventional pharmaceutical excipients, carriers or diluents.

[0034] The pharmaceutical composition also includes pharmaceutically acceptable excipients, including but not limited to fillers, lubricants, disintegrants, binders, glidants, etc.

[0035] In the preferred technical solution of the present invention, the pharmaceutically acceptable excipients include, but are not limited to, one or more of polyvinyl pyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methyl cellulose and its derivatives, ethyl cellulose and its derivatives, hydroxypropyl cellulose and its derivatives, hydroxypropyl methyl cellulose, starch and its derivatives, polyethylene glycol and its derivatives, lactose, lactose starch complex, lactose cellulose complex, sucrose, mannitol, mannitol starch complex, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, povidone, copovidone, calcium hydrogen phosphate, calcium stearate, sodium stearyl fumarate, silicon dioxide, titanium dioxide, talc, indigo, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, cross-linked polyvinyl ether, magnesium stearate, sodium stearyl fumarate, talc, and stearic acid, or a combination thereof.

[0036] In an optional embodiment, the above-mentioned medicine is a liquid pharmaceutical preparation (such as a kind of as an injection), for example, a solution, a suspension and a gel usually contain a liquid carrier, such as water and / or a pharmaceutically acceptable organic solvent. In addition, such liquid preparations may also include a pH adjusting agent, an emulsifier or a dispersant, a buffer, a preservative, a wetting agent, a gelling agent (such as methylcellulose), such as defined above. The medicine may be isotonic, that is, it may have an osmotic pressure identical to that of blood. The isotonicity of the medicine may be adjusted using sodium chloride and other pharmaceutically acceptable reagents, such as glucose, maltose, boric acid, sodium tartrate, propylene glycol and other inorganic or organic soluble substances. The viscosity of the liquid composition may be adjusted by a pharmaceutically acceptable thickening agent such as methylcellulose. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The preferred concentration of the thickening agent depends on the selected reagent.

[0037] In a preferred embodiment of the present invention, the dosage form of the drug is an injection.

[0038] In a second aspect, the present invention provides the use of a nucleic acid molecule, a recombinant vector or a gene editing system targeting a growth hormone receptor in the preparation of a drug for preventing or treating retinal neovascularization. The nucleic acid molecule targets the coding gene of the growth hormone receptor to perform gene knockout, site-directed base change, or site-directed insertion on the coding gene of the growth hormone receptor to achieve downregulation of the transcription level of the coding gene of the growth hormone receptor; the recombinant vector includes a nucleic acid molecule, and the gene editing system includes: a gene editing protein and a nucleic acid molecule, and the gene editing protein is selected from a Cas protein or a fusion of a Cas protein and a cytidine or adenosine deaminase.

[0039] In one embodiment, Cre-loxp gene knockout technology can also be used to achieve gene editing.

[0040] Cas proteins such as Cas9 protein, Cas12a or Cas13a Cytidine or adenosine deaminases are, for example, selected from CBEs or ABEs. Cytosine base editors (CBEs) can convert C·G to T·A, and adenine base editors (ABEs) can convert A·T to G·C. In the case of CBEs, a uracil glycosylase inhibitor (UGI) is often added to improve base editing efficiency and purity.

[0041] In a preferred embodiment of the present invention, the nucleic acid molecule is sgRNA or shRNA.

[0042] In a preferred embodiment of the present invention, the retinal neovascularization is selected from neonatal retinopathy, diabetic retinopathy, retinal vein occlusion or age-related macular degeneration.

[0043] In a preferred embodiment of the present invention, the above application includes at least one of the following methods: (1) Reduce the level of GHR in the subjects' retinal pericytes; (2) reduce the level of GHR-positive cells in the subjects' retina; (3) Slowing down the development of retinal blood vessels; (4) Reduce the number of deep retinal capillaries; (5) Reduce retinal pathological neovascularization; (6) Inhibit retinal vascular leakage and inflammation; (7) Reduce the area of retinal neovascularization; (8) Treatment of retinal neovascularization that is intolerant or unresponsive to VEGF antagonists.

[0044] In a preferred embodiment of the present invention, the drug further comprises pharmaceutically acceptable excipients, including but not limited to conventional pharmaceutical excipients, carriers or diluents.

[0045] The pharmaceutical composition also includes pharmaceutically acceptable excipients, including but not limited to fillers, lubricants, disintegrants, binders, glidants, etc.

[0046] In a third aspect, the present invention provides the use of a VEGF antagonist and a growth hormone receptor antagonist or the above-mentioned growth hormone receptor-targeting nucleic acid molecule, recombinant vector or gene editing system in the preparation of a drug for the combined treatment of retinal neovascular diseases.

[0047] VEGF antagonists include, but are not limited to, ranibizumab, bevacizumab, pegaptanib, and non-antibody VEGF antagonists such as aflibercept (VEGF-trap).

[0048] In a preferred embodiment of the present invention, the drug further comprises pharmaceutically acceptable excipients, including but not limited to conventional pharmaceutical excipients, carriers or diluents.

[0049] The pharmaceutical composition also includes pharmaceutically acceptable excipients, including but not limited to fillers, lubricants, disintegrants, binders, glidants, etc.

[0050] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0051] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0052] The present invention uses genetically engineered mice to discover the expression characteristics of GHR in the retina, laying the foundation for analyzing the function and role of retinal GHR. creERT2 ; GHR (fl / fl) and GHR creERT2 ;mT / mG; DTA mice, two different types of genetically engineered mice, conditionally knocked out retinal GHR, to observe changes in retinal vascular development; using PDGFR-β creERT2 ; GHR (fl / fl) and GHR creERT2 ;mT / mG; DTA mice, two different types of genetically engineered mice, were used to conditionally knock out retinal GHR in the classic OIR model (the OIR model can simulate retinal disease in premature infants) to observe the effects on pathological vascular changes; two different types of GHR receptor blockers, antisense oligonucleotide ATL1103 and GH analog GH-G120R, were directly injected into the vitreous of mice in the OIR model to observe the effects on pathological vascular changes; GHR was directly knocked out in the OIR model creERT2 mT / mG mice were injected with rhGH intravitreally to observe the effects of rhGH on pathological blood vessels and the changes in GHR during different intervention processes in the OIR model.

[0053] Example 1 This embodiment constructs GHR creERT2 ;mT / mG genetically engineered mice were used to analyze the expression characteristics of GHR in the retina, thus laying the foundation for analyzing the function and role of retinal GHR.

[0054] Building GHR creERT2 ;mT / mG genetically engineered mice (expressing GHR creERT2 and mT / mG genes). GHR creERT2 ;mT / mG mouse construction information reference Figure 1 As shown in A, GHR creERT2The mT / mG mice were obtained from Jicui Pharmaceuticals and were obtained from Jackson Laboratory, USA. To verify the GHR marker effect, the nonsteroidal anti-estrogen tamoxifen (concentration: 10 mg / mL) was used to induce GHR expression in cells at different time points. Mouse eyes were perfused and harvested (P7, P10, P14, P21, 1M, 4M, and 12M, where P represents day and M represents month) and fixed with 4% PFA for one hour at room temperature. Retinal flat mounts were then dissected and fluorescently stained. Endothelial cells and pericytes were labeled with antibodies to CD31 and PDGFRβ, respectively. The inner retina was imaged using SpinSR (Olympus) and data were analyzed using ImageJ and other software. mT / mG is a marker gene.

[0055] Results reference Figure 1 As shown, the results showed that GHR was mainly expressed in retinal pericytes ( Figure 1 B in the figure), with a small amount expressed in vascular endothelial cells, Müller cells, etc., and the overall expression level gradually increased over time ( Figure 1 C in the figure), it reaches its maximum at 1 month of age and remains relatively stable after adulthood.

[0056] Example 2 During the development of mouse retinal blood vessels, PDGFR-β creERT2 ; GHR (fl / fl) and GHR creERT2 ;mT / mG;DTA mice are two different types of genetically engineered mice, among which PDGFR-β creERT2 Genetic mice were obtained from Jackson Laboratory, GHR (fl / fl) gene mice and DTA gene mice were obtained from China Jicui Pharmaceutical Co., Ltd. The construction information is referenced from Figure 4 As shown. Knockout of retinal GHR and observe changes in retinal vascular development. DTA gene protein expression can kill cells expressing GHR. Retinal PDGFR-β (platelet-derived growth factor receptor β) is mainly expressed in pericytes. creERT2 ; GHR (fl / fl) mice can express Cre enzyme and carry the GHR (fl / fl) sequence. By inducing and activating Cre enzyme, the GHR DNA fragment between the two loxp sites can be specifically removed, blocking the transcription of the GHR gene.

[0057] Under normal physiological conditions, tamoxifen (concentration: 10 mg / mL) was used to treat GHR (fl / fl) of the littermate control group and PDGFR-β knockout group at different time points. creERT2 ; GHR (fl / fl) gene mice were induced, PDGFR-β creERT2; GHR (fl / fl) transgenic mice showed a conditional knockout effect of GHR in retinal pericytes (PDGFRβ marker). According to the experimental method of Example 1, retinal flat mounts of two groups of mice were fluorescently stained for CD31 to observe the changes in retinal vascular development; similarly, tamoxifen was used at different time points to treat the mT / mG; DTA of the littermate control group and the knockout group GHR. creERT2 ; mT / mG; DTA gene mice were induced, and the GHR knockout group creERT2 ; mT / mG; DTA gene mouse retinal GHR + The cells were specifically reduced, and the retinal flat mounts of mice in the two groups were fluorescently stained for CD31 to observe the changes in retinal vascular development.

[0058] Figure 5 The results showed that PDGFR-β creERT2 ; After GHR (fl / fl) mice were knocked out during the development of retinal pericytes, the deep (DL) and superficial (SL) vascular network areas at P10 were compared with those at Figure 5 As shown in Figure A, the deep (DL) and superficial (SL) vascular network areas are reduced.

[0059] GHR creERT2 ; mT / mG; After GHR was knocked out during retinal development in DTA mice, the changes in GHR expression and deep vascular network area were compared with those in Figure 5 As shown in Figure B, the results showed that the area of the deep (DL) and superficial (SL) vascular networks was reduced.

[0060] In summary, during the retinal vascular development stage of mice, both GHR knockout methods led to slower retinal vascular development, especially a significant reduction in deep retinal capillaries.

[0061] Example 3 Utilizing PDGFR-β creERT2 ; GHR (fl / fl) and GHR creERT2 ;mT / mG; DTA mice, two different types of genetically engineered mice, were used to knock out retinal GHR in the classic OIR model to observe the effects on pathological vascular changes.

[0062] Using the Tawang high oxygen chamber equipment, the mouse OIR model was established (refer to the model construction information diagram Figure 6 At P7, newborn mice were placed in a hyperoxia chamber and fed normally by their mothers, with the oxygen concentration maintained at 75%. At P12, they were taken out of the hyperoxia chamber and placed in an animal room at room temperature for further feeding. At P17, the mice were harvested and the model was completed. creERT2; GHR(fl / fl) transgenic mice), and retinal GHR + Cell knockout mice (littermates included control group mT / mG; DTA and knockout group GHR creERT2 ;mT / mG; DTA transgenic mice) were used to construct OIR models. Tamoxifen (concentration: 10 mg / mL) was used to induce OIR in both types of transgenic mice at P12, P13, P14, and P15 for 4 consecutive days. Retinal samples were collected at P17 to observe pathological vascular changes in the retinas of the model mice.

[0063] The expression characteristics of GHR in the retina of OIR model mice refer to Figure 7 shown.

[0064] Combine Figure 2 、 Figure 3 The results showed that in the OIR model, the conditional knockout of PDGFR-β creERT2 ; GHR (fl / fl) and GHR creERT2 ; mT / mG; After GHR knockout in the retina of DTA gene mice, neovascularization (NV) was significantly reduced.

[0065] Further investigation of conditional knockout mice with PDGFR-β creERT2 Fibrinogen was detected in GHR (fl / fl) mice. Figure 3 As shown, the results showed that in the OIR model, retinal vascular exudation was significantly reduced after knocking out GHR in the retina.

[0066] GHR knockout in both types of mice in the OIR model effectively reduced pathological neovascularization in the retina of P17 mice and controlled vascular leakage and inflammation compared with the control group.

[0067] Example 4 Two different types of GHR receptor blockers, ATL1103 (antisense oligonucleotide, MCE, Lot# 548214, Cat# HY-164419A) and GH-G120R (GH analog, MCE, Lot# 559990, Cat# HY-P7S0216), were directly injected into the vitreous of mice with OIR model to observe the effects on pathological vascular changes.

[0068] C57 mice and GHR creERT2 The OIR model was established in mT / mG mice according to the above method. Tamoxifen (concentration: 10 mg / mL) was used to treat GHR at P10, P11, P12, and P13. creERT2; mT / mG mice were induced and the model mice were intraocularly administrated at P14 time point. The control group mice were injected with 1ul solvent (sterile NaCl) into the right eye vitreous cavity; the experimental group mice were injected with 1ul GHR blocker (ATL1103 or GH-G120R) into the right eye vitreous cavity. The concentration of ATL1103 was 5mg / mL; the concentration of GH-G120R was 0.25mg / mL. The injection volume was 1ul for both groups. The specific intravitreal injection method is as follows: 20% urethane solution is used for anesthesia, with a dose of approximately 5 μl / g (depending on mouse body weight) based on body weight (can be reduced as appropriate). The palpebral fissure is carefully opened with microscissors, and a small amount of oxybuprocaine anesthetic is applied to the ocular surface. The mouse is positioned in lateral recumbency, and the right eye is exposed. A 34G Hamilton needle is used to aspirate the target agent, evacuate air bubbles, and then the needle is inserted at a 60-degree angle along the sclera approximately 1 mm proximal to the corneal limbus to a depth of approximately 1-2 mm. Approximately 1 μl of the agent is slowly introduced. No obvious bleeding or iris embolism is observed. After needle removal, levofloxacin ophthalmic gel is applied to the ocular surface, and the procedure is complete. At P17, the mouse eyeballs were perfused, sampled, retinal flat mounts were taken, confocal microscopy images were taken, and data analysis was performed as previously described.

[0069] Figure 8 The results suggest that intraocular administration of two GHR blockers, ATL1103 and GH-G120R, significantly reduced retinal neovascularization (NV) and can effectively improve the pathological changes of retinal neovascularization in the OIR model. Figure 8 The sham in the figure refers to the control group, i.e., NaCl was injected into the group.

[0070] Example 5 GHR in the OIR model creERT2 mT / mG mice were injected intravitreally or subcutaneously with rhGH to observe the effects of rhGH on pathological blood vessels and the important role of GHR in rhGH intervention in neovascularization in the OIR model.

[0071] Currently, there are no reports in the literature on the effects of direct rhGH treatment in the mouse OIR model. This study utilized various commercially available rhGH reagents (purchased from Novo Nordisk and Sezen) and conducted experimental observations using both local and systemic interventions. For local intervention, the experimental group of mice received a local injection of rhGH into the right vitreous cavity at P14 of the OIR model, while the control group received an equal volume of NaCl injection into the right vitreous cavity. The eyeballs were harvested at P17. For systemic intervention, the OIR model was treated with systemic rhGH. From P7 to P17, the experimental group received daily subcutaneous injections of 5 μg / g rhGH, while the control group received an equal volume of saline. The eyeballs were harvested at P17. Subsequent retinal flat mount staining and other procedures were performed as previously described, and vascular data were analyzed.

[0072] The results showed that after intraocular injection of rhGH on the 14th day (P14) of the OIR model, GHR expression increased, significantly aggravated the area of retinal neovascularization in P17 mice, and significantly increased retinal neovascularization (NV). Figure 9 A in the figure); After continuous subcutaneous injection of rhGH from day 7 (P7) to day 17 (P17) to intervene in the retinal blood vessels of mice, GHR expression also increased, significantly aggravating the area of retinal neovascularization in P17 mice ( Figure 9 B). This suggests that GHR plays a key role in rhGH-induced neovascular retinal changes. Figure 9 The sham group refers to the control group, i.e., the NaCl injection group. The rhGH group refers to the rhGH injection group.

[0073] In summary, the present invention constructs GHR creERT2 ;mT / mG genetically engineered mice, for the first time discovered the accurate expression location, morphology and other characteristics of GHR in the retina. The present invention uses different GHR knockout mice to clarify the key role of GHR in retinal vascular development. In the OIR model, different GHR knockout mice were also used to find that GHR has an important growth-promoting effect on retinal neovascularization; the use of different GHR blockers or antagonists has the same effect as that of GHR gene knockout mice, and all of them have the characteristics of rapid onset; blocking or inhibiting GHR can reduce pathological blood vessels while also reducing vascular exudation and inflammatory responses.

[0074] The invention provides a new direction for the treatment of retinal neovascular diseases and a new option for patients who are intolerant or unresponsive to anti-VEGF treatment.

[0075] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Use of a growth hormone receptor antagonist in the preparation of a drug for preventing or treating retinal neovascularization diseases.

2. The use according to claim 1, characterized in that The growth hormone receptor antagonist is selected from antisense oligonucleotides or GH analogs targeting growth hormone receptor mRNA, and the GH analog is selected from growth hormone mutants or pegvisomant; the growth hormone mutant is selected from at least one of the following: H18D, H21N, Q46K, F54P, R64K, G120K or G120R, R167N, K168A, D171S, K17R, E174S, F176Y and I179T, and the growth hormone mutant is a mutation based on the growth hormone amino acid sequence.

3. The use according to claim 2, characterized in that The antisense oligonucleotide is ATL1103.

4. The use according to claim 2, characterized in that The amino acid sequence of the growth hormone is shown in SEQ ID NO.

1.

5. The use according to claim 1, characterized in that The retinal neovascularization disease is selected from neonatal retinopathy, diabetic retinopathy, retinal vein occlusion or age-related macular degeneration; The application includes at least one of the following methods: (1) Reduce the level of GHR in the subjects' retinal pericytes; (2) reduce the level of GHR-positive cells in the subjects' retina; (3) Slowing down the development of retinal blood vessels; (4) Reduce the number of deep retinal capillaries; (5) Reduce retinal pathological neovascularization; (6) Inhibit retinal vascular leakage and inflammation; (7) Reduce the area of retinal neovascularization.

6. Use of a nucleic acid molecule, recombinant vector or gene editing system targeting growth hormone receptor in the preparation of a drug for preventing or treating retinal neovascularization, characterized in that: The nucleic acid molecule targets the gene encoding the growth hormone receptor to perform gene knockout, site-specific base change, and site-specific insertion on the gene encoding the growth hormone receptor to achieve downregulation of the transcription level of the gene encoding the growth hormone receptor; the recombinant vector includes the nucleic acid molecule, and the gene editing system includes: a gene editing protein and the nucleic acid molecule, and the gene editing protein is selected from Cas protein or a fusion of Cas protein and cytidine or adenosine deaminase.

7. The use according to claim 6, characterized in that The nucleic acid molecule is sgRNA or shRNA.

8. The use according to claim 6, characterized in that The retinal neovascularization is selected from neonatal retinopathy, diabetic retinopathy, retinal vein occlusion or age-related macular degeneration.

9. The use according to any one of claims 6 to 8, characterized in that: The application includes at least one of the following methods: (1) Reduce the level of GHR in the subjects' retinal pericytes; (2) reduce the level of GHR-positive cells in the subjects' retina; (3) Slowing down the development of retinal blood vessels; (4) Reduce the number of deep retinal capillaries; (5) Reduce retinal pathological neovascularization; (6) Inhibit retinal vascular leakage and inflammation; (7) Reduce the area of retinal neovascularization.

10. Use of a VEGF antagonist and a growth hormone receptor antagonist as described in any one of claims 1 to 5, or a growth hormone receptor-targeting nucleic acid molecule, recombinant vector, or gene editing system as described in any one of claims 6 to 9, in the preparation of a drug for the combined treatment of retinal neovascularization diseases.

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