CN110025768B - Construction method and application of animal model of eye diseases - Google Patents

Construction method and application of animal model of eye diseases Download PDF

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CN110025768B
CN110025768B CN201910476999.XA CN201910476999A CN110025768B CN 110025768 B CN110025768 B CN 110025768B CN 201910476999 A CN201910476999 A CN 201910476999A CN 110025768 B CN110025768 B CN 110025768B
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vegfr
iris
vegf
animal model
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CN110025768A (en
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刘堃
蒋炎
郑颖
王婧
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Shanghai First Peoples Hospital
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure

Abstract

The invention provides a construction method and application of an animal model of eye diseases, in particular to a molding reagent, which comprises the following steps: (a) a VEGF sustained release formulation; (b) VEGFR, or an expression vector thereof. The present invention can effectively construct an animal model of ocular diseases by using (a) a VEGF sustained-release preparation and (b) VEGFR, or an expression vector thereof in combination.

Description

Construction method and application of animal model of eye diseases
Technical Field
The invention relates to the technical field of biology, in particular to a construction method and application of an eye disease animal model.
Background
The formation of ocular neovascularization is one of the clinical causes of blindness in ophthalmology, and is mainly related to the angiogenic diseases causing ischemia and hypoxia in eyes. Diseases causing new blood vessels are many, and clinically common diseases comprise diabetes, retinal artery obstruction, ischemic central retinal vein obstruction and the like. Neovascularization may occur in many tissues of the eye, such as corneal neovascularization, iris neovascularization, choroidal neovascularization, ciliary neovascularization, and retinal neovascularization. Therefore, the study of key molecules that influence their production, or the discovery of new drugs that interfere with their progression, will have a major impact on the progression of ophthalmic clinics.
Iris neovascularization of the iris (NVI), also known as rubeosis, is often secondary to diseases in other parts of the eye and systemic diseases. Studies have shown that iris neovascularization occurs at 1% -17% in the diabetic population, and up to 65% in proliferative diabetic retinopathy. Drugs for treating retinal neovascularization, such as combaiccept, aflibercept, bevacizumab and ranibizumab, are widely used clinically, and research on intervention of generation and development of corneal neovascularization by novel biological peptide is successful at first, but research on mechanism and treatment of iris neovascularization is poor. One of the challenges facing NVI-related studies is the lack of an ideal animal model. At present, a photochemical method is mostly adopted for establishing a classical animal NVI model, and the main principle is that a photochemical drug rose bengal is injected into an animal vein, and then the animal eye is irradiated by laser to excite oxygen in blood to be converted into oxygen free radicals. The excited oxygen radicals destroy vascular endothelial cells and platelet membranes, and then start the blood coagulation system. At the same time, the broken platelets release a series of clotting factors, accelerating thrombus formation. In this model, rabbits, cats, pigs, monkeys, rats, etc. are often used as experimental animals. However, although the large and medium-sized animals have large eyeballs, the large and medium-sized animals are not suitable for large-scale use due to high cost, and this is a disadvantage that the model is not negligible.
Therefore, there is an urgent need in the art to provide new experimental models for animals.
Disclosure of Invention
The invention aims to provide a novel animal experimental model.
The invention provides in a first aspect a moulding agent comprising:
(a) A VEGF sustained release formulation;
(b) VEGFR, or an expression vector thereof.
In another preferred embodiment, the VEGF sustained release formulation is selected from the group consisting of: VEGF microparticles, VEGF suspensions, or combinations thereof.
In another preferred embodiment, the VEGF sustained release formulation comprises VEGF microsomes.
In another preferred embodiment, the expression vector comprises a viral vector.
In another preferred embodiment, the viral vector is selected from the group consisting of: an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, or a combination thereof.
In another preferred embodiment, the microparticles comprise nanoparticles.
In another preferred embodiment, the diameter of the microparticles is 30 to 300nm, preferably 50 to 200nm, more preferably 100 to 150nm.
In another preferred embodiment, the total drug loading of the microparticles is from 2 to 50. Mu.g, preferably from 5 to 40. Mu.g, more preferably from 6 to 20, more preferably from 8 to 15. Mu.g.
In another preferred embodiment, the drug concentration of the microsomes in the molding agent is 20-90 ng/. Mu.l, preferably 30-70 ng/. Mu.l, more preferably 40-55 ng/. Mu.l.
In another preferred embodiment, the molding agent is a liquid preparation.
In another preferred embodiment, the molding agent is a suspension of nanoparticles.
In another preferred embodiment, the modeling agent consists essentially of (. Gtoreq.90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) or all of (a) a VEGF-delaying agent and (b) VEGFR, or an expression vector thereof.
In another preferred embodiment, the concentration of component (a) in the molding agent is 20 ng/. Mu.l to 100 ng/. Mu.l, preferably 30 ng/. Mu.l to 60 ng/. Mu.l, more preferably 50 ng/. Mu.l to 55 ng/. Mu.l.
In another preferred embodiment, the concentration of component (b) in the molding agent is 1X 10 7 mu.l-6X 10 7 Mu.l, preferably 2X 10 7 mu.l-5X 10 7 Mu.l, more preferably, 3X 10 7 mu.l-3.5X 10 7 Mu.l/l.
In another preferred embodiment, the VEGFR protein comprises a full-length protein or a protein fragment.
In another preferred embodiment, the VEGFR protein is derived from a mammal, more preferably a rodent (e.g., mouse, rat), primate, and human.
In another preferred embodiment, the VEGFR protein further comprises a derivative of a VEGFR protein.
In another preferred embodiment, the derivatives of the VEGFR proteins include modified VEGFR proteins, protein molecules having amino acid sequences homologous to native VEGFR proteins and having VEGFR protein activity, dimers or multimers of VEGFR proteins, fusion proteins containing VEGFR protein amino acid sequences.
In another preferred embodiment, the expression "a protein molecule having an amino acid sequence homologous to a native VEGFR protein and having native VEGFR protein activity" means that the amino acid sequence has greater than or equal to 85% homology, preferably greater than or equal to 90% homology, more preferably greater than or equal to 95% homology, and most preferably greater than or equal to 98% homology to the VEGFR protein; and protein molecules having native VEGFR protein activity.
In another preferred embodiment, the molding agent is an agent for preparing an animal model of an ocular disease.
In another preferred embodiment, the animal model comprises an animal model of a non-human mammal.
In another preferred embodiment, the ocular disease comprises an iris neovascular related disease.
In another preferred embodiment, the iris neovascular related disease is selected from the group consisting of: retinal vein occlusion with neovascular glaucoma, diabetic retinopathy with neovascular glaucoma, or a combination thereof.
In a second aspect, the invention provides a use of the modeling agent of the first aspect of the invention in the preparation of a medicament or a formulation for an animal model of an ocular disease.
In another preferred embodiment, the formulation is a liquid formulation.
In another preferred embodiment, the ocular disease comprises an iris neovascular related disease.
In another preferred embodiment, the iris neovascular related disease is selected from the group consisting of: retinal vein occlusion with neovascular glaucoma, diabetic retinopathy with neovascular glaucoma, or a combination thereof.
The third aspect of the present invention provides a method for preparing an animal model of an ocular disease, comprising:
(a) Providing a mammal;
(b) Injecting the first component and the second component into the ocular tissue of the mammal, and culturing for a period of time T1, thereby obtaining the animal model of the ocular disease.
In another preferred embodiment, the ocular tissue is selected from the group consisting of: the anterior chamber, the iris, or a combination thereof.
In another preferred embodiment, the first component comprises a VEGF sustained release formulation.
In another preferred embodiment, the second component comprises VEGFR, or an expression vector thereof.
In another preferred embodiment, the first component and the second component are injected sequentially or simultaneously.
In another preferred embodiment, when the first component and the second component are injected sequentially, the interval between the injection time of the first component and the injection time of the second component is 12h to 6 days, preferably 1 day to 4 days, and more preferably 3 days to 4 days.
In another preferred embodiment, T1 is 7-21 days, preferably 10-18 days, more preferably 14-16 days.
In another preferred embodiment, the mammal comprises a human or non-human mammal.
In another preferred embodiment, the non-human mammal includes a rodent (e.g., rat, mouse, rabbit), a primate (e.g., monkey).
In another preferred embodiment, the aqueous humor of the anterior chamber is contained in a volume of 1. Mu.l to 10. Mu.l, preferably 1. Mu.l to 5. Mu.l, and more preferably 3. Mu.l to 5. Mu.l.
In another preferred embodiment, the concentration of said first component is between 10 ng/. Mu.l and 100 ng/. Mu.l, preferably between 30 ng/. Mu.l and 60 ng/. Mu.l, more preferably between 50 ng/. Mu.l and 55 ng/. Mu.l.
In another preferred embodiment, the concentration of the second component is 1 × 10 7 mu.l-6X 10 7 Mu.l, preferably 2X 10 7 mu.l-5X 10 7 Mu.l, more preferably, 3X 10 7 mu.l-3.5X 10 7 Mu.l/l.
In a fourth aspect, the invention provides the use of an animal model prepared by the method of the third aspect of the invention as an animal model for studying ocular diseases.
In a fifth aspect, the invention provides the use of an animal model prepared by the method of the third aspect of the invention to screen for or identify substances (therapeutics) that can alleviate or treat an ocular disease.
In a sixth aspect, the present invention provides a method of screening or identifying potential therapeutic agents for treating or ameliorating an ocular disorder, comprising the steps of:
(a) Administering a test compound to the animal model prepared by the method of claim 3 in the presence of the test compound in a test group, and determining the severity of ocular disease Q1 in the animal model in the test group; and detecting the severity of ocular disease Q2 of the animal model in a control group that is not administered the test compound and is otherwise identical; and
(b) Comparing the severity Q1 and severity Q2 detected in the previous step to determine whether the test compound is a potential therapeutic agent for treating or ameliorating an ocular disease;
wherein, if severity Q1 is significantly lower than severity Q2, it is indicative that the test compound is a potential therapeutic agent for treating or ameliorating an ocular disease.
In another preferred embodiment, said detecting the severity of ocular disease comprises detecting a change in one or more indicators selected from the group consisting of: increased intraocular pressure, whether the iris tissue has visible new blood vessels under the lens, and whether the iris angiography has fluorescence leakage.
In another preferred embodiment, the reduced severity of ocular disease is characterized by: intraocular pressure drop, regression of neovasculature under the iris histoscope, and reduction of fluorescence leakage in iris angiography.
In another preferred embodiment, the phrase "significantly less than" means that the ratio of the severity Q1/severity Q2 is less than or equal to 1/2, preferably less than or equal to 1/3, and more preferably less than or equal to 1/4.
In another preferred embodiment, the method is non-diagnostic and therapeutic.
In another preferred embodiment, the method comprises the step of (c) administering the potential therapeutic agent screened or identified in step (b) to an animal model prepared by the method of the third aspect of the invention, thereby determining its effect on the severity of ocular disease in said animal model.
In a seventh aspect, the invention provides a non-human mammalian model prepared by a method according to the third aspect of the invention.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows VEGFR overexpression in tissue samples of this study.
FIG. 2 shows the intraocular pressure in rats of each group of the study.
FIG. 3 shows the anterior segment of the eye after modeling in the experimental mouse of this study.
Figure 4 shows the results of the iris angiography after modeling of the experimental mice of this study.
FIG. 5 shows the change in intraocular pressure after injection of 10nm diameter and 500nm VEGF microsomes in the experimental mice of this study.
FIG. 6 shows the change in intraocular pressure of experimental mice in this study after injection of VEGF microsomes at a drug concentration of 10 ng/. Mu.l.
Detailed Description
The present inventors have conducted extensive and intensive studies and have unexpectedly found that an animal model of ocular diseases can be efficiently constructed by using (a) a VEGF sustained-release preparation and (b) VEGFR, or an expression vector thereof in combination. Furthermore, the present inventors have also surprisingly found that both (a) a VEGF sustained release preparation and (b) VEGFR or an expression vector thereof can be injected into an ocular tissue (e.g., anterior chamber) sequentially or simultaneously to effectively construct an animal model of an ocular disease, and that the animal model of the present invention can be used for the study of ocular diseases and for the screening and testing of specific drugs. The present invention has been completed based on this finding.
Specifically, the present inventors constructed VEGFR overexpressing Adenoviruses (ADV) and VEGF microsomes and injected the VEGFR overexpressing adenoviruses in the anterior chamber of rats. After the over-expression is confirmed, iris neogenesis blood vessels and retina blood vessels of the experimental mouse are sealed by laser, and VEGF microsomes are injected into the anterior chamber of the experimental mouse which is sealed by the laser. Detecting the intraocular pressure of the experimental mouse, and observing side reaction and new blood vessels by iris angiography of a front section camera. Successful overexpression in rat iris tissue after intracameral injection of VEGFR adenovirus takes 48-72 hours, so we injected VEGF microsomes 3 days after intracameral injection of adenovirus in rats. VEGF has a short biological half-life, lasts only 4 to 6 hours even under hypoxic conditions, requires repeated injections to maintain the effective concentration required for model construction, and is prone to ocular complications. Therefore, the present invention uses nanoparticles to construct a VEGF sustained release system to solve the above existing problems. In addition, adenovirus can be expressed for 1 week after being transfected successfully in rat iris tissue, therefore, the invention constructs VEGF microsome with the diameter of about 150nm, and the release period is about 10 days, thereby maintaining the effective concentration of model construction and delaying the resolution of NVI to the maximum extent.
Iris Neovascularization (NVI)
As used herein, the term "NVI" refers to iris neovascularization, also known as rubeosis, which refers to the pathological growth of blood vessels on the surface of iris tissue under conditions of ischemia and hypoxia. NVI is not a primary disease of the iris, but is secondary to many eye diseases and some systemic diseases. Because it can develop into or combine with the formation of fiber vascular membrane, the iris-corneal angle is closed to cause serious neovascular glaucoma, the intraocular pressure is often difficult to control, and finally the eye is blinded, even the eyeball is removed due to severe pain. Therefore, it is important to find and handle as early as possible. Early in NVI, first stage, new blood vessels first appear in the near pupillary edge of the iris and in certain areas of the angle of the atrium. The surface of the iris was seen as a fine curved and irregular red line on a brown iris. The width of the chamber angle is still normal when the iris-cornea angle is checked. The duration of this period varies with the cause of the disease, and the progression is rapid due to central retinal vein occlusion, which lasts for only weeks or months. In the second stage, iris neovessels continue to increase and fuse with each other until the neovessels on the whole iris surface become reticular, and the iris corneal angle also has more neovessels but no or only a few areas of iris peripheral anterior synechia. NVI progresses to stage III, where the iris surface is generally obscured by neovascular membranes; due to the contraction of fiber vascular tissues, the pigment layer is pulled forward to form the extraversion of the pupil edge pigment layer; extensive peripheral anterior synechia of the iridocorneal angle results in a dramatic increase in intraocular pressure and a significant mixed hyperemia of neovascular glaucoma. The eyes are severely painful, and the vision only shows light sensation. Grading NVI of the model mouse by adopting a Miller grading method, wherein the NVI is divided into 0-5 grades, and the 0 grade is a normal iris, a small amount of blood vessels are visible or invisible, and no leakage exists; grade 1, visible blood vessels increase, protrude, meander, are discontinuous, but still have no leakage; 2-3 grades, i.e. blood vessels increase, and early and rapid new blood vessel leakage occurs; level 4 NVI was so abundant that the iris was not visible within the first 35s, and level 5: 4 combined with iris eversion or glaucoma.
In the present invention, the diseases associated with the neovascularisation of the iris include, but are not limited to: retinal vein occlusion with neovascular glaucoma, diabetic retinopathy with neovascular glaucoma.
VEGF sustained release formulations
In the present invention, the VEGF sustained-release preparation refers to nanoparticles or suspensions containing VEGF, the half-life of which can be maintained for only 4-6 hours even under anoxic conditions, and the VEGF sustained-release preparation is used to allow the VEGF to be slowly and continuously released so as to maintain an effective concentration required for model construction.
VEGFR proteins and polynucleotides
In the present invention, the terms "protein of the invention", "VEGFR protein", "VEGFR polypeptide" are used interchangeably and all refer to a protein or polypeptide having the amino acid sequence of VEGFR. These include VEGFR proteins with or without an initiating methionine. In addition, the term also includes full-length VEGFRs and fragments thereof. The VEGFR proteins of the present invention include their complete amino acid sequences, their secreted proteins, their mutants and functionally active fragments.
VEGFR proteins are vascular endothelial growth factor receptors that bind to multiple growth factors of the VEGF family, thereby inducing a range of angiogenic responses.
In the present invention, the terms "VEGFR gene", "VEGFR polynucleotide" are used interchangeably and refer to a nucleic acid sequence having a VEGFR nucleotide sequence.
The genome of the human VEGFR gene has a full length of 5849bp (NCBI GenBank accession NC-000004.12).
The genome of rat VEGFR gene has a total length of 5892bp (NCBI GenBank accession NC-005113.4).
It is understood that nucleotide substitutions in codons are acceptable when encoding the same amino acid. It is also understood that nucleotide changes are also acceptable when conservative amino acid substitutions are made by nucleotide substitutions.
When an amino acid fragment of VEGFR is obtained, a nucleic acid sequence encoding it can be constructed therefrom, and a specific probe can be designed based on the nucleotide sequence. The full-length nucleotide sequence or a fragment thereof can be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers may be designed based on the VEGFR nucleotide sequences disclosed herein, particularly the open reading frame sequences, and the relevant sequences may be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.
Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into cells, and isolating the relevant sequence from the propagated host cells by conventional methods.
In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.
At present, DNA sequences encoding the proteins of the present invention (or fragments, derivatives thereof) can be obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art.
The polynucleotide sequences of the present invention may be used to express or produce recombinant VEGFR polypeptides by conventional recombinant DNA techniques. Generally, the following steps are performed:
(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a human VEGFR polypeptide, or with a recombinant expression vector comprising the polynucleotide;
(2) A host cell cultured in a suitable medium;
(3) Isolating and purifying the protein from the culture medium or the cells.
In the present invention, the VEGFR polynucleotide sequence may be inserted into a recombinant expression vector. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translation control elements.
Methods well known to those skilled in the art can be used to construct expression vectors containing VEGFR-encoding DNA sequences and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits 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 E.coli.
Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, bacterial cells of the genus streptomyces; fungal cells such as yeast; a plant cell; an insect cell; animal cells, and the like.
Transformation of host cells with recombinant DNA the present invention may be usedBy conventional techniques well known to those skilled in the art. When the host is prokaryotic, such as E.coli, competent cells, which are capable of DNA uptake, can be harvested after exponential growth phase and subsequently treated with CaCl 2 Methods, the steps used are well known in the art. Another method is to use MgCl 2 . If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.
The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.
The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated 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 such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.
Adenoviral vectors
Adenovirus (ADV) is a particle without an envelope and with a diameter of 70-90 nm, and is composed of 252 capsomeres arranged in a icosahedron shape. The diameter of each shell particle is 7-9 nm. Inside the capsid is a linear double-stranded DNA molecule of about 4.7kb with inverted repeats of about 100bp each at both ends. Since the 5' end of each DNA strand has a relative molecular mass of 55X10 3 Da protein molecules are covalently bound, and a circular structure of double-stranded DNA can be formed.
There are 52 known human adenoviruses, named adl-ad 52, and ad2 has been studied in the most detail. Transcription of the adenovirus genome produces mRNA, known as a transcription unit of at least 5: the EI region is located at the left side of the viral genome and can be subdivided into EIA and EIB which are related to cell transformation; the EI region encodes a DNA binding protein involved in viral replication; the EIII region encodes a glycoprotein which is present on the surface of the host cell; the EIV region is positioned at the right end of the ad2 genome and is regulated and controlled by DNA binding protein coded by the EII region; the 5 th transcription unit synthesizes the ad2 protein IV in the middle of viral infection.
Adenoviruses are carcinogenic to rodents, or can transform rodent cells cultured in vitro. Only a portion of the adenovirus genome is required for cell transformation, and these genes are located at the left end of the genome and constitute approximately 7% to 10% of the entire genome. Although the adenovirus is widely distributed, the adenovirus does not have carcinogenicity to human bodies. Human cells are permissive cells (permissive cells) that allow replication of invading viruses upon infection, and eventually cell lysis and death to release large amounts of progeny virus. Adenovirus particles are not detected in many human tumor cells cultured in vitro, but there is an integration site for adl2 on human chromosome 1, which means that human cells may also be nonpermissive for adenovirus, i.e., such cells cannot replicate in cells after viral infection, but can integrate within the genome of infected cells. These cells are transformed with viruses, the phenotype is altered, and can be cultured for passage in vitro for indefinite periods.
Adeno-associated virus
Due to the characteristics of Adeno-associated virus (AAV) that it is smaller than other viral vectors, it is not pathogenic, and can transfect dividing and non-dividing cells, gene therapy methods for genetic diseases based on AAV vectors have received much attention.
Adeno-associated virus (AAV), also called adeno-associated virus, belongs to the genus dependovirus of the family parvoviridae, is the simplest single-stranded DNA-deficient virus of the currently discovered class, and requires a helper virus (usually adenovirus) to participate in replication. It encodes the cap and rep genes in inverted repeats (ITRs) at both ends. ITRs are crucial for replication and packaging of viruses. The cap gene encodes the viral capsid protein, and the rep gene is involved in viral replication and integration. AAV can infect a variety of cells.
The recombinant adeno-associated virus (rAAV) is derived from non-pathogenic wild adeno-associated virus, is considered to be one of the most promising gene transfer vectors due to the characteristics of good safety, wide host cell range (divided and non-divided cells), low immunogenicity, long time for expressing foreign genes in vivo and the like, and is widely applied to gene therapy and vaccine research in the world. Over 10 years of research, the biological properties of recombinant adeno-associated viruses have been well understood, and many data have been accumulated on the application effects of recombinant adeno-associated viruses in various cells, tissues and in vivo experiments. In medical research, rAAV is used in the study of gene therapy for a variety of diseases (including in vivo, in vitro experiments); meanwhile, the gene transfer vector is used as a characteristic gene transfer vector and is widely applied to the aspects of gene function research, disease model construction, gene knock-out mouse preparation and the like.
In a preferred embodiment of the invention, the vector is a recombinant AAV vector. AAV is a relatively small DNA virus that can integrate into the genome of cells that they infect in a stable and site-specific manner. They are able to infect a large series of cells without any effect on cell growth, morphology or differentiation and they do not appear to be involved in human pathology. AAV genomes have been cloned, sequenced and characterized. AAV contains an Inverted Terminal Repeat (ITR) region of about 145 bases at each end, which serves as the viral origin of replication. The remainder of the genome is divided into two important regions with encapsidation functions: the left part of the genome comprising the rep gene involved in viral replication and viral gene expression; and the right part of the genome comprising the cap gene encoding the viral capsid protein.
AAV vectors can be prepared using standard methods in the art. Any serotype of adeno-associated virus is suitable. Methods for purifying the carrier can be found, for example, in U.S. patent nos. 6566118, 6989264 and 6995006, 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 disclosure of which is incorporated herein by reference in its entirety. The use of vectors derived from AAV for in vitro and in vivo gene transfer has been described (see, e.g., international patent application publication Nos. WO91/18088 and WO93/09239; U.S. Pat. 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 a gene of interest, and the use of these constructs to transport the gene of interest in vitro (into cultured cells) or in vivo (directly into the organism). Replication-defective recombinant AAV can be prepared by co-transfecting the following plasmids into a cell line infected with a human helper virus (e.g., adenovirus): plasmids containing the nucleic acid sequence of interest flanked by two AAV Inverted Terminal Repeat (ITR) regions, and plasmids carrying AAV encapsidation genes (rep and cap genes). The AAV recombinants produced are then purified by standard techniques.
In some embodiments, the recombinant vector is encapsidated into a virion (e.g., an AAV virion including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV 16). Accordingly, the disclosure includes recombinant viral particles (recombinant as they comprise a recombinant polynucleotide) comprising any of the vectors described herein. Methods of producing such particles are known in the art and are described in U.S. Pat. No.6,596,535.
Molding reagent
The invention also provides a molding agent, which comprises effective dose of (a) VEGF slow-release preparation; and (b) a VEGFR, or an expression vector thereof.
Generally, (a) a VEGF sustained release formulation; and (b) VEGFR, or an expression vector thereof, in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, such as physiological saline, at a pH of generally about 5 to about 8, preferably at a pH of about 7 to about 8.
The term "effective amount" or "effective as used hereinDosage "refers to the amount of adenovirus that is sufficiently expressed in iris tissue after transfection and does not cause death of the animal; the amount of microparticles capable of sustained release of VEGF after injection was stable (please supplement). In a preferred embodiment of the present invention, the effective amounts are: VEGF microsome, 10 ng/. Mu.l-100 ng/. Mu.l, preferably 30 ng/. Mu.l-60 ng/. Mu.l, more preferably 50 ng/. Mu.l-55 ng/. Mu.l. VEGFR adenovirus, 1X 10 7 mu.l-6X 10 7 Mu.l, preferably 2X 10 7 mu.l-5X 10 7 Mu.l, more preferably, 3X 10 7 mu.l-3.5X 10 7 Mu.l/l. Preferably, the effective amount is administered in a single injection.
As used herein, a "pharmaceutically acceptable" component is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier, including for administration of a therapeutic agent. The pharmaceutically acceptable carrier which may be used in the present invention is not particularly limited and may be one or more compatible solid or liquid fillers or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of intermixing with the adipose mesenchymal progenitor cells of the present invention without significantly diminishing their therapeutic effectiveness. Examples of pharmaceutically acceptable carrier moieties of the invention are physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions, suitable aqueous and nonaqueous carriers, diluents, solvents or excipients including water, ethanol, polyols and suitable mixtures thereof. In addition to the conventional vectors described above, optimized vectors can also be designed based on the properties of the adipose mesenchymal progenitor cells. The carrier is preferably an infusion solution carrier and/or an injection carrier.
The modeling agent of the invention contains an effective amount of (a) a VEGF sustained-release preparation; and (b) a VEGFR, or an expression vector thereof. The pharmaceutical preparations should generally be adapted to the mode of administration, and the pharmaceutical molding agents of the present invention may be prepared in the form of injection preparations, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The molding agent of the invention can also be prepared into a sustained-release preparation.
The molding agent of the present invention is preferably an injection preparation, more preferably an intracameral puncture injection preparation. In another preferred embodiment, the concentration of the VEGF slow release preparation in the modeling reagent (such as the reagent for paracentesis injection) is 10ng/μ l to 100ng/μ l, preferably 30ng/μ l to 60ng/μ l, more preferably 50ng/μ l to 55ng/μ l, and the concentration of the expression vector of VEGFR is 1X 10 7 mu.l-6X 10 7 Mu.l, preferably 2X 10 7 mu.l-5X 10 7 Mu.l, more preferably, 3X 10 7 mu.l-3.5X 10 7 Mu.l/l. The injection mode of the molding agent is not particularly limited, and the molding agent can be a single injection preparation or a combination of multiple injections. In a preferred embodiment of the present invention, the molding agent is a single injection.
In the invention, the molding agent is preferably an injection preparation, and more preferably an intracameral puncture injection preparation.
Animal model
In the present invention, a very effective non-human mammalian model for studying ocular diseases is provided.
In the present invention, examples of non-human mammals include (but are not limited to): mouse, rat, rabbit, monkey, etc., more preferably rat and mouse.
Drug candidate or therapeutic agent
In the present invention, there is also provided a method of screening for a candidate drug or therapeutic agent for alleviating or treating an ocular disease using the animal model of the invention.
In the present invention, a drug candidate or therapeutic agent refers to a substance known to have a certain pharmacological activity or being tested, which may have a certain pharmacological activity, including but not limited to nucleic acids, proteins, chemically synthesized small or large molecular compounds, cells, and the like. The candidate drug or therapeutic agent may be administered orally, intravenously, intraperitoneally, subcutaneously, or intradermally.
The main advantages of the invention include:
(a) The invention discovers for the first time that the combination of (a) a VEGF slow-release preparation and (b) VEGFR or an expression vector thereof can effectively construct an animal model of eye diseases.
(b) The invention discovers for the first time that the animal model of the eye diseases can be effectively constructed by injecting (a) the VEGF slow-release preparation and (b) the VEGFR or the expression vector thereof into the eye tissues (such as the anterior chamber) sequentially or simultaneously.
(c) The invention discovers for the first time that the combination of (a) VEGF slow-release preparation and (b) VEGFR or expression vector thereof can effectively construct animal model of eye diseases, and compared with the classical model building method of iris neovascularization (laser closed retina blood vessel), the side effects of anterior segment of eye (corneal edema, aqueous humor flare, blood aqueous humor, etc.) are obviously reduced.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
The materials used in the examples are all commercially available products unless otherwise specified.
EXAMPLE 1 Experimental murine overexpression of VEGFR in the eyes
Rat anterior chamber injection of VEGFR overexpression adenovirus (3.5X 10) 7 One/. Mu.l), 3 days later, iris tissues of the experimental mice were randomly extracted from the normal control group, the ADV-VEGFR group and the ADV-blank vector group, subjected to Western blot experiment, and subjected to differential analysis of protein expression.
The results are shown in FIG. 1. FIG. 1 shows the protein expression and statistics of VEGFR in the tissue samples of the present invention, and the expression of VEGFR in the ADV-VEGFR group was significantly up-regulated (P < 0.05) compared to the ADV-blank vector group, and there was no difference in VEGFR expression between the ADV-blank vector group and the blank control group (P > 0.05).
Example 2 intraocular pressure monitoring results analysis
After verifying VEGFR overexpression, VEGF microsomes (50 ng/. Mu.l, total microsome loading 10. Mu.g, diameter 150 nm) were injected into the anterior chamber, and the effect of nanoparticles on the aqueous outflow tract was excluded with blank microsomes. Intraocular pressure was monitored by tonometer in each group of rats at the same time period daily and statistical analysis of intraocular pressure results was performed.
The results are shown in FIG. 2. FIG. 2 shows that at day 7 after molding, the intraocular pressure of rats in VEGF microparticle group starts to increase (14.97. + -. 0.9207 mmHg), the intraocular pressure at day 14 is obviously increased (24.23. + -. 0.9333 mmHg) compared with that of the blank microparticle group, and the intraocular pressure drops to a normal level (9.533. + -. 0.4055 mmHg) at day 21; the intraocular pressure of rats in the blank control group, the ADV-empty vector injection group, the ADV-VEGFR injection group and the blank particle group is unchanged.
Example 3 analysis of photographic results of previous section
Observing anterior segment change by slit lamps at 1, 2, 4,7, 14 and 21 days after model building, injecting 10% chloral hydrate into abdominal cavity to anaesthetize rat, and paying attention to ciliary hyperemia, corneal edema, corneal posterior deposition, aqueous humor clouding flash or bloody aqueous humor, iris tissue color change, whether the iris tissue has visible neovascularization under the mirror, and pupil shape change.
The results are shown in FIG. 3. FIG. 3 shows that no side effects such as corneal edema, corneal postcorneal pigmentation, aqueous humor flare, etc. appear in the anterior segment of the experimental mouse after the model is made.
Example 4 Iris angiography results analysis
After the model is made, the iris fluorescence radiography is carried out on days 7, 14 and 21, the anesthetized rat is injected with 10 percent chloral hydrate abdominal cavity, the injection is finished within l to 3s after the injection of 0.04ml of 20 percent fluorescein sodium injection through the tail vein, and the photographing is started at the same time until the fluorescence is subsided.
The results are shown in FIG. 4. FIG. 4 shows that VEGFR adenovirus (3.5X 10) was overexpressed by laser occlusion of iris and retinal vessels at day 14 post-molding 7 μ l) injection + VEGF microparticlesIn the body (concentration of 50 ng/. Mu.l, total drug loading of 10ul, diameter of 150 nm) group rat iris can see a large amount of fluorescence leakage and iris angiogenesis.
Example 5
Injection concentration in rat anterior chamber was 3.5X 10 7 A total of 5. Mu.l of ADV-VEGFR per μ l, 50ng/μ l of ADV-VEGFR at a total dose of 10ul after 3 days, and 5. Mu.l of VEGF microsomes with a diameter of 150nm. Iris angiography is performed on rats 14 days after model creation, the laser is used for sealing iris and retinal vessels, VEGFR adenovirus injection and VEGF microsome group rat irises are subjected to combined overexpression, a large amount of fluorescence leakage can be seen, and iris neovascularization is confirmed. The results are shown in FIG. 4.
Comparative example 1
The procedure is as in example 5, except that VEGF alone is injected.
The result shows that the intraocular pressure of the rat is not increased, no obvious fluorescein leakage is seen in the iris angiography, and the result shows that the iris neovascularization animal model is not successfully constructed.
Comparative example 2
The method was the same as in example 5, except that VEGFR or its expression vector alone was injected.
The result shows that adenovirus transfection in rat iris tissues is successful, VEGFR is successfully over-expressed, but rat intraocular pressure is not increased, no obvious fluorescein leakage is seen in iris angiography, and the result shows that the iris neovascularization animal model is not successfully constructed.
Comparative example 3
The procedure is as in example 5, except that the injected microsomes are blank nanoparticules (without VEGF).
The result shows that the intraocular pressure of the rat is not increased, no obvious fluorescein leakage is seen in the iris angiography, and the result shows that the iris neovascularization animal model is not successfully constructed. The results are shown in FIG. 4.
Comparative example 4
The procedure was the same as in example 5 except that VEGF microsomes were injected at 10nm and 500nm in diameter, respectively.
The result shows that the intraocular pressure of the rat is not increased, no obvious fluorescein leakage is seen in the iris angiography, and the result shows that the iris angiogenesis animal model is not successfully constructed. The diameter of the microsome is too small, and the microsome can flow out of the anterior chamber along with aqueous humor through an aqueous humor outflow tract and cannot release VEGF into the anterior chamber; too large a diameter results in too slow release of VEGF, making it difficult to achieve effective concentrations of VEGF over an effective period of time. The results are shown in FIG. 5.
Comparative example 5
The method was the same as in example 5 except that VEGF microsome drug concentrations were 10 ng/. Mu.l and 100 ng/. Mu.l, respectively, for injection.
The result shows that the intraocular pressure of the rat is not increased, no obvious fluorescein leakage is seen in the iris angiography, and the result shows that the iris neovascularization animal model is not successfully constructed. The medicine concentration is too low to reach the effective concentration of angiogenesis, and the angiogenesis cannot be induced; the drug concentration is too high to exceed the VEGF tolerance of animals, and experimental animals die in large numbers. The results are shown in FIG. 6.
Discussion of the related Art
Neovascular glaucoma (NVG) is refractory glaucoma secondary to extensive retinal ischemia, is characterized by neovessels in the iris and the angle of the house, is a serious blinding eye disease, and is difficult to treat clinically and has poor curative effect. The formation of NVG is a dynamic development process, and the formation of NVI is assumed as a precondition, so that the establishment of an ideal animal model has important significance for the deep research of NVG, and the key point for the establishment of the NVG model lies in the establishment of the NVI model.
The traditional NVI model construction method mainly comprises a retinal vein occlusion model, a diabetes model, an oxygen-induced retinopathy model, an anterior segment ischemia model, a crystal cortex reinjection model, a transgenic mouse overexpression insulin-like growth factor-1 model and the like. The traditional model construction methods have the defects of limited animal sources, high cost, complex operation, more complications and the like, and are often difficult to successfully construct the NVI model.
As the research progresses, the present inventors have found that the effects of VEGF in neovasculature are not insignificant. In the eye aqueous humor of NVI patients, the content of VEGF is obviously increased, and the content of VEGF in the aqueous humor is closely related to the proliferation degree of the neovascularization on the surface of the iris. Therefore, the present invention utilizes the method of over-expression of VEGFR in combination with the anterior chamber injection of VEGF microparticles to construct NVI animal models. The NVI model constructed by the method has a series of advantages of simple and feasible manufacturing method, early occurrence time, controllable duration, small side reaction, high success rate and the like, and has certain challenge and novelty compared with the traditional NVI model. In addition, the NVI is constructed by the method, the formation mechanism of the NVI is clear and single, and the method can be used for research on the NVI formation induced by different growth factors, inflammatory cytokines and adhesion molecules. Conditions are created for researching related intervention in early NVG, and a platform is provided for screening related drugs.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (3)

1. A molding agent, comprising:
(a) A VEGF sustained release preparation which is VEGF microsome;
(b) VEGFR, or an expression vector thereof;
the molding reagent is used for preparing an iris neovascular disease animal model, the concentration of the component (a) in the molding reagent is 40 ng/mu l-55 ng/mu l, the diameter of the microsome is 100-150 nm, and the concentration of the component (b) in the molding reagent is 3 multiplied by 10 7 mu.l-3.5X 10 7 Mu.l/l.
2. Use of the molding agent of claim 1 for the preparation of a medicament for an animal model of iris neovascular disease.
3. Use of a molding agent according to claim 1 for the preparation of an animal model of iris neovascular disease.
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