Application of liver Sdhaf4 gene in retinopathy analysis or retinopathy model preparation

Abstract

The invention provides an application of a liver Sdhaf4 gene in preparation of a retinopathy model. The inventor is devoted to research on retinopathy for a long time, through deep research tests, a brand-new retinopathy-causing gene target of peripheral tissues related to retinopathy, namely the Sdhaf4 gene is disclosed, and through reasonable design, the Sdhaf4 gene can be used for preparing a new retinopathy-causing gene target. The retinopathy animal model under the condition of good blood glucose control can be obtained by targeting the target and implementing down-regulation. The animal model is stable and controllable in state, typical in disease phenotype and easy to observe.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine; more specifically, this invention relates to the application of the liver Sdhaf4 gene in the analysis of retinal lesions or the preparation of retinal lesion models. Background Technology

[0002] Retinopathy, one of the most common complications of diabetes, is a leading cause of blindness and severe visual impairment in the working population. Early symptoms of retinopathy include decreased visual function and increased retinal inflammation. In advanced stages, retinal neovascularization occurs, jeopardizing vision. Current treatments primarily include anti-VEGF therapy and laser therapy, but these are mainly for patients with advanced vision loss and their effectiveness is not ideal. Therefore, early diagnostic indicators and interventional targets are urgently needed. Early-stage patients can reduce their risk of visual impairment by controlling their blood sugar, but clinically, we still encounter cases where visual impairment occurs even with good blood sugar control.

[0003] While treatment for diabetic retinopathy is based on blood sugar control, its core lies in specific interventions targeting the eye lesions, which differs significantly from conventional diabetes treatment. The treatment of diabetic retinopathy is a dual-track system of "blood sugar management + targeted ophthalmic intervention," and these two approaches are not interchangeable. Once diabetes is diagnosed, annual fundus screening should be initiated as early as possible; early intervention can reduce the risk of blindness.

[0004] Clinically available interventional treatments for diabetic retinopathy include, for example, laser photocoagulation (local photocoagulation: sealing leaking microvessels (treatment of macular edema)) and vitrectomy (removing vitreous hemorrhage and repositioning the retina (late-stage complication)).

[0005] Clinically available specific treatments for retinopathy (not required for conventional diabetes) also include anti-VEGF drug therapy, which involves directly injecting ranibizumab / aflibercept into the vitreous cavity to inhibit angiogenesis and reduce macular edema.

[0006] Given that the core of diagnosing or treating retinopathy lies in specific interventions targeting the eye lesions and that the diagnostic or treatment protocols for diabetes cannot be applied directly, there is an urgent need in this field to further explore the influencing factors related to retinopathy in order to find new clinical prevention and treatment approaches. Summary of the Invention

[0007] The purpose of this invention is to provide the application of the liver Sdhaf4 gene in the preparation of a retinal disease model.

[0008] In a first aspect of the invention, a method for preparing an animal (model) of retinopathy (associated) is provided, comprising downregulating the Sdhaf4 gene in the liver of the animal.

[0009] In one or more embodiments, the downregulation targets regions of exons 1, 2, and / or 3 of the liver Sdhaf4 gene.

[0010] In one or more embodiments, the downregulation targets the region of exon 2 and / or exon 1 of the liver Sdhaf4 gene.

[0011] In one or more embodiments, the animal is a rodent; preferably, it includes mice, rats, hamsters, and rabbits; more preferably, it is a mouse.

[0012] In one or more embodiments, the rodents may also include animals selected from the group consisting of: porcupines, guinea pigs, capybaras, beavers, marmots, and squirrels.

[0013] In one or more embodiments, the retinopathy (related) animal is an animal with normal blood glucose levels but retinal lesions.

[0014] In one or more embodiments, the downregulation includes: specifically knocking out the liver Sdhaf4 gene using Cre-loxP-mediated recombination.

[0015] In one or more embodiments, a loxP strategy is used to target the Sdhaf4 locus, with a pair of loxP sites flanking exons 1, 2, and / or 3, thereby deleting the exons after Cre-loxP-mediated recombination.

[0016] In one or more embodiments, animals with a pair of loxP sites (one on each side) flanking exons 1, 2 and / or 3 of the Sdhaf4 locus in their genome are hybridized with Alb-Cre animals to specifically knock out the liver Sdhaf4 gene.

[0017] In one or more embodiments, the downregulation includes: infecting an animal with a virus carrying a Sdhaf4 interfering molecule; preferably, the Sdhaf4 interfering molecule is shRNA; more preferably, the shRNA is shRNA with a sense strand CGUCUGGAGAGCAGCAAGATT and an antisense strand UCUUGCUGCUCUCCAGACGTT.

[0018] In one or more embodiments, the animal model of retinopathy exhibits a phenotype selected from the following: activation of retinal microglia, increased number of migrating cells, and migration from the inner periphery (IPL) to the inner nuclear layer (INL); a significant increase in the positive area of ​​Iba-1 labeling; and increased levels of retinal inflammatory factors. Preferably, the retinal inflammatory factors include: IL-1b, IL6, IL8, IL18, TNF-α, and HIF1α; and increased levels of CD11b protein, a molecular marker of microglia.

[0019] In one or more embodiments, the knockout of liver Sdhaf4 forms homozygous Sdhaf4. - / - .

[0020] In one or more embodiments, the knockout of the liver's Sdhaf4 results in a homozygous liver-specific knockout.

[0021] In another aspect of the invention, the application of the animal model of retinopathy prepared by the method is provided for: serving as an animal model for screening candidate drugs or therapeutic agents for the treatment of retinopathy; serving as an animal model for studying retinopathy; or for conducting drug metabolism and toxicology tests.

[0022] In one or more embodiments, the method or application for preparing an animal model of retinopathy or for preparing animal cells is a non-diagnostic or non-therapeutic method or application, and is not intended for the direct purpose of diagnosing or treating the disease.

[0023] In another aspect of the invention, the use of a reagent that downregulates the liver Sdhaf4 gene in an animal in a kit for preparing an animal model of retinopathy is provided; wherein the reagent is selected from: Sdhaf4 interfering molecules; reagents that specifically knock out the liver Sdhaf4 gene.

[0024] In one or more embodiments, the Sdhaf4 interfering molecule is shRNA; more preferably, the shRNA is shRNA with a sense strand CGUCUGGAGAGCAGCAAGATT and an antisense strand UCUUGCUGCUCUCCAGACGTT.

[0025] In one or more embodiments, the reagent for specifically knocking out the liver Sdhaf4 gene is a Cre-loxP-mediated recombination method for specifically knocking out the liver Sdhaf4 gene.

[0026] In one or more embodiments, the reagent targets exons 1, 2, and / or 3 of the Sdhaf4 locus.

[0027] In another aspect of the present invention, a method for screening candidate drugs or therapeutic agents for treating retinopathy is provided, the method comprising: (1) Use the method described above to prepare an animal model of retinal disease; (2) The candidate substance is given to the animal model of (1) and the candidate substance is observed to have a therapeutic effect on retinopathy. If the symptoms of retinopathy in the animal model are observed to be improved, the candidate substance is an animal substance for treating or alleviating retinopathy.

[0028] In one or more embodiments, during the observation of the method for screening candidate drugs or therapeutic agents for treating retinopathy, the transcriptional levels of retinal inflammatory factors IL-1β, IL6, IL8, IL18, TNF-α and / or HIF1α in the animal model are analyzed; wherein, if the levels of one or more inflammatory factors decrease significantly, the candidate substance is an animal for treating or alleviating retinopathy.

[0029] In one or more embodiments, during the observation of the method for screening candidate drugs or therapeutic agents for treating retinopathy, the protein expression of microglia molecular markers CD11b and IL-1β is analyzed; if the protein expression of CD11b and IL-1β is significantly reduced, then the candidate substance is an animal for treating or alleviating retinopathy.

[0030] In one or more embodiments, during the observation of the method for screening candidate drugs or therapeutic agents for treating retinopathy, visual function is analyzed by electroretinography. If the b-wave is significantly increased and visual function is enhanced, then the candidate substance is suitable for treating or alleviating retinopathy in animals.

[0031] In one or more embodiments, the observation of the method for screening candidate drugs or therapeutic agents for treating retinopathy is carried out by analyzing the Iba-1 immunofluorescence positive areas of the retina. If the positive area of ​​Iba-1 labeling is significantly reduced, the candidate substance is an animal for treating or alleviating retinopathy.

[0032] In one or more embodiments, the test may include setting up control animals that are not given the candidate substance.

[0033] In one or more embodiments, the candidate drug includes known or newly developed: compounds (e.g., compounds derived from or modified from public compound libraries); biomacromolecules (proteins (e.g., binding molecules such as antibodies or ligands), nucleic acids (e.g., nucleic acid inhibitors, interfering molecules, etc.)), etc.

[0034] In one or more embodiments, the method further includes: conducting further cell experiments and / or animal experiments on the obtained potential substances to further select and identify substances useful for treating retinopathy from the candidate substances.

[0035] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0036] Figure 1 Patients with retinopathy have a SNP in SDHAF4, which is located in exon 2 of the SDHAF4 gene.

[0037] Figure 2 The efficiency of Sdhaf4 knockdown in AAV-Sdhaf4 shRNA mice.

[0038] Figure 3 Mice with AAV-Sdhaf4 shRNA had normal blood glucose levels.

[0039] Figure 4 AAV-Sdhaf4 shRNA activates mouse retinal microglia.

[0040] Figure 5 AAV-Sdhaf4 shRNA increased the levels of inflammatory factors in the mouse retina.

[0041] Figure 6 The knockout efficiency of Sdhaf4 in liver-specific knockout Sdhaf4 mice.

[0042] Figure 7 Blood glucose levels were normal in liver-specific knockout Sdhaf4 mice.

[0043] Figure 8 Activation of retinal microglia in liver-specific knockout Sdhaf4 mice.

[0044] Figure 9 Liver-specific knockout Sdhaf4 mice showed increased levels of retinal inflammatory factors.

[0045] Figure 10 Liver-specific knockout of Shaf4 mice resulted in decreased visual function. Detailed Implementation

[0046] The inventors have long been dedicated to the research of retinopathy. Through in-depth research and experimentation, they have revealed a novel gene target associated with retinopathy, namely the Sdhaf4 gene. By rationally designing a model, targeting and downregulating this target can generate animal models of retinopathy. These animal models are stable and controllable, exhibit typical disease phenotypes, and are easy to observe.

[0047] Current research suggests that Sdhaf4 is associated with aging and cardiovascular disease. However, there are no studies reported on the function of Sdhaf4 in patients with retinal diseases and animal models, and it is unknown whether it is related to inflammatory markers in the early stages of retinal disease.

[0048] the term

[0049] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art.

[0050] As used in this invention, the animals referred to are rodents; preferably including: mice, rats, hamsters, rabbits, etc.

[0051] As used herein, “sgRNA” refers to “single-guide RNA” or “single guide RNA”, which is designed based on “target sites on target genes”. The sequence it contains is sufficient to work synergistically with endonucleases Cas (such as Cas9) to guide Cas enzyme-mediated DNA double-strand breaks at the target sites.

[0052] As used in this invention, the term "sgRNA target" refers to a target region in the gene of interest suitable for gene editing. For example, regulation can be achieved by introducing exogenous gene editing reagents (such as sgRNA and CassmRNA) targeting this target.

[0053] As used herein, "exogenous" or "heterogeneous" refers to the relationship between two or more nucleic acid or protein sequences from different sources, or the relationship between cells and hosts from different sources. For example, if the combination of nucleic acid and host cell is not naturally occurring, the nucleic acid is heterologous to that host cell. A particular sequence is "heterogeneous" to the cell or organism into which it is inserted.

[0054] As used herein, the terms “introduction” or “transformation” refer to the transfer of exogenous polynucleotides into a host (an animal or animal cell in this invention).

[0055] As used herein, the term "construction" includes "plasmid".

[0056] Animal models and applications

[0057] Retinal inflammation is an early pathological feature of retinopathy, and microglia activation is a major contributor to retinal inflammation and a primary source of IL-1β in the retina. This study found that while complete Sdhaf4 knockout animals were embryonic lethal, AAV-shRNA-mediated liver Sdhaf4 knockout animals, with normal liver function and blood glucose levels indistinguishable from control animals, showed increased retinal microglia activation and retinal inflammation levels after one month of AAV intervention. Furthermore, liver-specific Sdhaf4 knockout animals initiated by Albumin-Cre, with normal liver function and blood glucose levels indistinguishable from control animals, showed increased retinal microglia activation, retinal inflammation levels, and visual impairment.

[0058] Robust disease phenotypes are crucial for developing new treatments because they provide sufficient capacity to evaluate drug efficacy. However, identifying suitable genes as regulatory targets for retinopathy remains challenging, and establishing successful retinopathy models in non-human mammals has been difficult to achieve to date. Retinopathy is a disease with independent symptoms, and the core of its treatment lies in specific interventions targeting the ocular lesions. Furthermore, to determine the effectiveness of treatments and ensure the successful translation of these new therapies into clinical applications, it is necessary to create animal models that are more similar to humans in terms of genes, physiological structure, and disease phenotype.

[0059] In their preliminary research, the inventors conducted detailed analyses targeting multiple targets and aspects, ultimately identifying the liver's Sdhaf4 gene as a target. By downregulating this target, an animal model of retinal disease was obtained. In a preferred embodiment, the inventors further combined Cre-loxP-mediated recombination technology or RNA interference technology to prepare the animal model.

[0060] Targeting a suitable liver Sthaf4 gene site can yield better results. Case analysis of clinical samples showed that patients with omental lesions exhibited a SNP in Sthaf4. This SNP is located in exon 2 of the Sthaf4 gene, with a mutation from genotype A to G (A / G polymorphism). This indicates that the SNP is closely associated with disease progression in patients, resulting in superior animal phenotypic performance. Therefore, exon 2 was identified as a preferred target for targeted therapy.

[0061] In this invention, gene downregulation includes "knockout," "deletion / deficiency," "inactivation," or "suppression," meaning that the gene or the protein it encodes is not produced, or is produced in the host cell in an inactive form, or is produced in the host cell at a level lower than that found in the wild-type form in the host cell under the same or similar growth conditions.

[0062] Under the guidance of this invention, various gene-disrupting methods known in the art can be employed to target the gene targets identified in this invention. These can be achieved, for example (but not limited to), through one or more of the following methods: CRISPR / Cas technology, homologous recombination, RNA interference-based techniques, ZFN, and TALEN, etc.

[0063] As another preferred approach, homologous recombination can be used to specifically target the Sdhaf4 gene, causing defective or absent expression. Alternatively, Cre and LoxP methods can be applied to selectively knock out, reduce, or inactivate related genes in the cell genome.

[0064] As another optional approach of this invention, a CRISPR / Cas (such as Cas9) system can be used for targeted gene editing, thereby modifying the Sdhaf4 gene in the target region. Common knockout methods include co-transferring sgRNA or nucleic acids that can form said sgRNA, Cas mRNA or nucleic acids that can form said Cas mRNA to the target region or target cells. After identifying the target site, known methods can be used to introduce sgRNA and Cas into the cells. The CRISPR / Cas can be CRISPR / Cas9 technology. This includes introducing a specific sgRNA targeting the Sdhaf4 gene into an animal, while simultaneously introducing Cas9 mRNA to facilitate gene editing. After identifying the target site, sgRNA or nucleic acids that can form said sgRNA, Cas9 mRNA or nucleic acids that can form said Cas9 mRNA are co-transferred into animal zygotes to obtain gene-edited animals. Alternatively, the nucleic acid capable of forming the sgRNA can be a nucleic acid construct or expression vector, or the nucleic acid capable of forming the Cas9 mRNA can be a nucleic acid construct or expression vector. These expression vectors are introduced into cells, thereby forming active sgRNA and Cas9 mRNA within the cells. Furthermore, Cas9 mRNA and sgRNA can also be obtained through in vitro transcription.

[0065] Constructing gene knockout animals is an important technique in biomedical research, with several main purposes: (1) Studying gene function: By knocking out (i.e., completely removing or inactivating) a specific gene, researchers can observe the effects of this change on the animal's physiology and behavior, thereby inferring the role and function of the gene in the organism. This helps to reveal how genes control the development of organisms, physiological processes, and the occurrence of diseases. (2) Simulating human diseases: Many human diseases are related to variations or dysfunctions of specific genes. By knocking out these same genes in animals, researchers can create animal models that simulate human genetic diseases. These models are crucial for studying the mechanisms of disease development, pathological processes, and the development of new treatments. (3) Drug development and testing: Gene knockout animals are widely used in drug development, especially in the early stages of pharmacodynamics and toxicology research. By testing new drugs on these animal models, researchers can assess the safety, side effects, and effectiveness of drugs in a specific gene context. (4) Understanding the interaction between genetic and environmental factors: Gene knockout animals can also be used to study how the interaction between genetic factors and environmental factors (such as diet, lifestyle, chemical exposure, etc.) affects the risk of health and disease. (5) Gene therapy research: By studying gene knockout animals, scientists can better understand which genes are potential gene therapy targets, and thus develop gene therapy strategies for hereditary diseases. In summary, gene knockout animals are a powerful tool for understanding the role of genes in organism development, physiological function, and disease.

[0066] While the construction of gene knockout animals provides a powerful tool for biomedical research, it also faces a series of challenges and difficulties: (1) Technical complexity: Although gene knockout technology, especially gene editing technology using CRISPR-Cas9, is relatively mature, it still requires highly specialized knowledge and skills. The correct design of guide RNA (gRNA), ensuring the precise editing of the target gene, and subsequent genotyping steps all require precise operation and strict control. (2) Off-target effects: Off-target effects may occur during gene editing, that is, the editing tool may not only act on the target gene, but may also accidentally modify the DNA sequence of other non-target sites. This non-specific editing may lead to unexpected genetic variations, affecting the accuracy of experimental results and the health of animals. (3) Gene redundancy and compensation mechanisms: Some genes may have functional redundancy, that is, when a gene is knocked out, other genes with similar functions may compensate for its function, which makes it difficult to analyze the direct effects of a single gene knockout. (4) Embryo lethality: Some genes are crucial for embryonic development, and their knockout may lead to early embryonic lethality, making it impossible to obtain adult gene knockout animals, thus limiting the study of the function of these genes. (5) Complexity of phenotypic analysis: Even after successfully constructing gene knockout animals, analyzing their phenotypes (i.e., the physiological and behavioral changes caused by gene knockout) is a complex process. Gene knockout may affect multiple physiological processes, and these effects may be influenced by various factors such as environmental factors, age, and sex. Despite these challenges, this invention has selected suitable targets and obtained a successful gene knockout animal model.

[0067] The animal models constructed in this invention can be used for screening and testing specific drugs. In drug screening, candidate drugs or therapeutics refer to substances known to have certain pharmacological activities or substances currently being tested that may have certain pharmacological activities, including but not limited to nucleic acids, proteins, carbohydrates, chemically synthesized small or large molecular compounds, and cells. The administration routes for candidate drugs or therapeutics can be oral, intravenous, intraperitoneal, subcutaneous, spinal, or direct intracerebral injection.

[0068] As those skilled in the art will understand, due to the complexity of the organism's genes, the influence of multiple signaling pathways on diseases, and the existence of the body's own compensatory or repair mechanisms, it is difficult to obtain animal models that exhibit typical disease symptoms. However, this invention, through optimized design, overcomes these technical difficulties. The animal model constructed by this invention can serve as a powerful tool for scientific research and new drug evaluation.

[0069] In the study of disease mechanisms, the animal model constructed in this invention, which stably presents retinopathy, can be used to investigate the disease mechanism, explore the key factors leading to retinopathy caused by Sdhaf4 protein deficiency, and investigate the intermediate mechanisms that can prevent or delay the development of this disease. The model system of this invention helps to better understand retinopathy and to explore / identify candidate drugs / therapeutic agents that can prevent, delay, or reverse the disease process.

[0070] In preclinical drug testing in vivo, the animal model constructed using this invention, which stably presents retinopathy, is expected to be used for preclinical drug metabolism, toxicity, and efficacy testing. This animal model is physiologically close to the human body and supports long-term sampling, detection, and tracking, facilitating the advancement of new drug development. In this invention, there are no particular limitations on the types of candidate drugs used for drug testing; they can be obtained from various sources, including synthetic or natural compound libraries. For example, there are various methods for the random and directed synthesis of various organic compounds and biomolecules, including the expression of random oligonucleotides and oligopeptides; or, natural compound libraries in the form of bacterial, fungal, plant, and animal extracts can be obtained or readily generated. Furthermore, libraries and compounds generated by natural or synthetic methods can be readily modified by conventional chemical, physical, and biochemical methods and can be used to generate combinatorial libraries. Known pharmacological reagents can be chemically modified (e.g., acylation, alkylation, esterification, amidation, etc.) to generate structural analogs.

[0071] The method for preparing animal models in this invention is simple to operate, and the resulting animal models are highly stable, effectively mimicking the phenotype of retinopathy. The resulting disease symptoms are very typical, and phenotypic changes are easily observed. The animal models of this invention provide a new approach for studying the pathogenesis of retinopathy, drug screening, and clinical treatment.

[0072] Based on the method of the present invention, the present invention also provides a kit for preparing an animal model of retinopathy, the kit comprising: a reagent for downregulating the liver Sdhaf4 gene of an animal, the reagent being selected from: a Sdhaf4 interfering molecule, or a reagent for specifically knocking out the liver Sdhaf4 gene; preferably, the Sdhaf4 interfering molecule is shRNA; more preferably, the shRNA is a shRNA with a sense strand CGUCUGGAGAGCAGCAAGATT and an antisense strand UCUUGCUGCUCUCCAGACGTT. Preferably, the reagent is a Cre-loxP-mediated recombination method for specifically knocking out the liver Sdhaf4 gene; more preferably, the reagent targets exons 1, 2, and / or 3 of the Sdhaf4 gene locus.

[0073] Other reagents commonly used in transgenic operations may also be included in the kit for the convenience of those skilled in the art, such as reagents for microinjection. Furthermore, the kit may include instructions for use by those skilled in the art.

[0074] Once an animal model of the present invention is obtained, substances of interest that can (or potentially can) treat retinopathy can be screened based on this model. Following screening, truly useful drugs can be identified from the substances of interest.

[0075] Therefore, the present invention also provides a method for screening potential substances, the method comprising: (1) preparing an animal model of retinal lesions using the method or kit described above; (2) administering a candidate substance to the animal model of (1) and observing whether the candidate substance has a therapeutic effect on retinal lesions; if the retinal lesion symptoms of the animal model are observed to be improved, then the candidate substance is an animal substance for treating retinal lesions.

[0076] In a preferred embodiment of the present invention, during screening, a control group may be set up to make it easier to observe changes in the symptoms of the retinal lesions. The control group may be the animal model without the addition of the candidate substance.

[0077] As a preferred embodiment of the invention, the method further includes: conducting further cell experiments and / or animal experiments and / or clinical trials on the obtained potential substances to further select and identify truly useful substances.

[0078] On the other hand, the present invention also provides potential substances of interest obtained using the aforementioned screening method. These initially screened substances can form a screening library, from which relatively ideal, safe, and truly effective substances for treating retinal diseases can ultimately be selected.

[0079] This invention overcomes the current lack of animal model tools for studying the major clinical challenge of developing retinopathy despite normal blood glucose levels. Based on the target provided by this invention and combined with existing knowledge in the field, simple and effective analytical methods and their preferred primers can be designed to establish stable and reproducible preclinical animal models.

[0080] The invention will be better understood from the following examples. However, those skilled in the art will understand that the specific methods and results are merely for illustrating the invention and not for limiting it. Experimental methods in the following examples that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Science Press, or according to the manufacturer's recommendations.

[0081] Sequence information

[0082] Mouse Sdhaf4 gene exon sequence (CDS sequence in uppercase): Exon 1 (SEQ ID NO: 1):

[0083] Exon 2 (SEQ ID NO: 2):

[0084] Exon 3 (SEQ ID NO: 3):

[0085] Human Sdhaf4 gene exon sequence (CDS sequence in uppercase): Exon 1 (SEQ ID NO: 4):

[0086] Exon 2 (SEQ ID NO: 5):

[0087] Exon 3 (SEQ ID NO: 6):

[0088] Example 1: Analysis of patients with retinal diseases

[0089] 1. Preparation of blood samples

[0090] In this embodiment, blood samples were obtained from patients with retinopathy (DR) in clinical settings.

[0091] 2. DNA extraction

[0092] Commercially available DNA extraction kits were used, and the procedures were followed according to the instructions.

[0093] 3. DNA quality testing

[0094] Take 5 μL of DNA solution and electrophoresis with 1% agarose and 1X TAE buffer (voltage 120-180 V). A single band indicates that the DNA is intact and has not been degraded, and a clear band indicates that the concentration can meet the requirements of PCR.

[0095] The concentration and purity were measured using a spectrophotometer. A 1 μl sample was taken to check the OD value. An OD 260 / 280 ratio between 1.7 and 2.0 indicates good DNA quality. A ratio less than 1.7 indicates protein contamination, and a ratio greater than 2.0 indicates RNA contamination. Generally, small amounts of protein and RNA contamination do not affect routine PCR.

[0096] 4. Primer design

[0097] Primer design software used is Primer Premier 5. Typical parameters are: Primer length 18–30 bp; Tm value 55-65 degrees, annealing temperature around 60 degrees; GC content 40-70%; Avoid the presence of primer dimers and nonspecific amplification; Avoid four consecutive bases, especially G and C. Ideally, there should not be more than three G or C bases in the last five bases at the 3' end. PCR amplification product length: site sequencing primers are about 150-300 bp long and 80-150 bp long from the site; exon detection primers are about 150 bp long upstream and downstream of the exon; the PCR product band of the target gene sequencing is generally no more than 1200 bp.

[0098] Primer sequence for SDHAF4: 1-F: 5'-TTTCACTCTGCGTCCTTTTGTAC-3' (SEQ ID NO: 7); 1-R: 5'-CGATGGTCAATATGGTGATTTTAC-3' (SEQ ID NO: 8).

[0099] 5. PCR reaction system

[0100] 6. PCR reaction conditions

[0101] 7. Electrophoretic detection bands

[0102] Take 5 μl of PCR product and perform electrophoresis on a 1% agarose gel. Electrophoresis parameters: 150 V, 100 mA, 10–20 min for observation.

[0103] 8. Purification and recovery of PCR products

[0104] The objective was to extract and recover PCR bands using gel cutting, following the method described in the SanPrep Column DNA Gel Extraction Kit (Sangon Biotech B518131).

[0105] 10. Data Analysis

[0106] Sequence Analysis software was used for sequence analysis, and SeqMan software was used for sequence alignment.

[0107] 11. Results Analysis

[0108] Detection of DNA amplification products extracted from blood samples of patients with retinopathy revealed the presence of a SNP in the SDHAF4 gene. This SNP is located in exon 2 of the SDHAF4 gene and is a mutation from genotype A to G (A / G polymorphism). The presence of the SDHAF4 SNP in blood samples from patients with retinopathy and its location detection results are as follows: Figure 1 As shown.

[0109] Based on the above, blood samples were collected from patients with well-controlled blood glucose but still developing retinopathy. Genomic analysis revealed a SNP in exon 2 of SDHAF4, indicating a close association between SDHAF4 and disease progression in these patients.

[0110] Example 2: Animal model of liver-specific knockdown

[0111] Sdhaf4 knockdown animals were prepared by targeting and knocking down SDHAF4.

[0112] 1. Preparation of liver-specific knockdown animals via AAV injection

[0113] C57BL / 6J mice were placed in a specialized restraint device. The tail was heated with 50°C warm water for 3-5 minutes to fully dilate the tail vein, and then disinfected with a 75% alcohol swab. Using a 1 ml insulin syringe, with the needle bevel facing upwards, the syringe was inserted into the skin at approximately a 20° angle to the tail, parallel to the vein. Once blood return was observed, the needle placement was confirmed to be correct. 100 μl of AF-shRNA-AAV or NC-shRNA-AAV virus solution was slowly and evenly injected. After injection, the needle was quickly withdrawn, and the injection site was pressed with a sterile dry cotton ball for 1 minute to stop bleeding.

[0114] The shRNA sequences targeting Sdhaf4(AF) are shown in Table 1.

[0115] Table 1

[0116] Analysis results of Sdhaf4 knockdown efficiency in AAV-Sdhaf4 shRNA animals are as follows: Figure 2 As shown, animals with successfully knocked-down Sdhaf4 were obtained.

[0117] Blood glucose levels in animals inoculated with AAV-Sdhaf4 shRNA were analyzed and compared with those in the control group. Figure 3 As shown, the blood glucose levels of the AAV-Sdhaf4 shRNA animals were normal.

[0118] Example 3: Pathological analysis of AAV-Sdhaf4 shRNA in animals

[0119] 1. Morphological analysis of retinal microglia

[0120] We analyzed the microglia in the retina of mice induced by AAV-Sdhaf4 shRNA and compared them with controls.

[0121] Experimental group: AAV-Sdhaf4 shRNA mice.

[0122] Control group: NC-shRNA-AAV mice.

[0123] The detection was performed using immunofluorescence staining, including: Iba-1 (green): Specific marker for microglia (resident immune cells in the retina). By observing the intensity, morphology, and location of the green signal, the state of microglia (such as whether they are activated, whether their morphology has changed, etc.) can be determined.

[0124] DAPI (blue): Marks the nuclei of all cells to show the overall hierarchical structure of the retina (GCL, IPL, INL, OPL, ONL).

[0125] Merge: Superimposes two fluorescence signals to precisely locate the specific positions of microglia in different layers of the retina.

[0126] The results showed that AAV-Sdhaf4 shRNA activated mouse retinal microglia, such as... Figure 4 Activation of retinal microglia is a key step in the pathogenesis of eye diseases, and abnormal activation is a typical manifestation of aggravated retinal damage.

[0127] 2. Analysis of inflammatory factors

[0128] The levels of inflammatory factors in the mouse retina were analyzed using AAV-Sdhaf4 shRNA. These inflammatory factors included IL-1β, IL-6, IL-8, IL-18, TNF-α, and HIF1α. Primers used for the analysis are shown in Table 2.

[0129] Table 2

[0130] like Figure 5 The results showed that in the mouse retina inoculated with AAV-Sdhaf4 shRNA, the levels of the inflammatory cytokine IL-8 and IL-1β were significantly increased, and the levels of IL-6 also showed a noticeable increase. Protein level analysis also showed a significant increase in the expression level of the inflammatory cytokine IL-1β.

[0131] Example 4: Preparation of Sdhaf4 knockout animals

[0132] According to the aforementioned Example 1, patients with retinal lesions have SNPs in exon 2. In this case, Sdhaf4 knockout animals were prepared by targeting and knocking out exon 2.

[0133] The creation of liver gene knockout mice: Sdhaf4 flox mice were bred by Biocytogen Ltd. (Beijing, China). The loxP strategy was used to target the Sdhaf4 gene locus to generate Sdhaf4-deficient mice. A pair of loxP sites were flanking exon 2 and were deleted after Cre-loxP-mediated recombination. Sdhaf4 flox mice were amplified by PCR and detected by direct sequencing. This was further confirmed by southern blotting.

[0134] Alb-Cre mice were obtained from Jackson Laboratory (Bar Harbor, ME, no. 003574).

[0135] The Sdhaf4 liver homozygous knockout mouse (Sdhaf4 Alb-KO) is produced by crossing Alb-Cre mice (Ravenclaw) with Sdhaf4 flox / flox mice. Sdhaf4 Alb-KO mice are born at the expected Mendelian ratio and exhibit normal fertility.

[0136] The results of the analysis of the Sdhaf4 knockout efficiency in liver-specific Sdhaf4 knockout animals are as follows: Figure 6 As shown, animals with Sdhaf4 knockout were successfully obtained.

[0137] Blood glucose levels were analyzed in liver-specific knockout Sdhaf4 animals and compared with those in the control group. Figure 7 As shown, the blood glucose levels of the AAV-Sdhaf4 shRNA animals were normal.

[0138] Example 5: Lesion analysis in animals with liver-specific knockout of Sdhaf4

[0139] 1. Morphological analysis of retinal microglia

[0140] Excess retrobulbar tissue was removed from the mouse eyeball under a stereomicroscope, taking care to preserve the optic nerve. 400 μl of 4% PFA was added to a 48-well plate, and the dissected eyeball was immersed in 4% PFA and fixed at room temperature for 15 min. The anterior segment and lens were removed, taking care to preserve the limbus. The dissected eyeball was then fixed in 4% PFA at room temperature for 30 min, washed with PBS for 10 min, and then successively dehydrated with 15% sucrose at room temperature for 30 min, followed by dehydration with 30% sucrose at room temperature for 30 min. The sucrose on the optic cup was blotted dry with absorbent paper, embedded in OCT, and then flash-frozen in liquid nitrogen. After freezing solid, frozen sections were prepared at a thickness of 10 μm and stored at -80°C.

[0141] After being removed from -80°C, retinal frozen sections were equilibrated at room temperature for 15 min. The sections were then washed three times with PBS for 5 min each to remove OCT. An immunohistochemical pen was used to circle the tissue to prevent leaching. The sections were placed horizontally in a humidified chamber and blocked at room temperature for 60 min with blocking buffer. After discarding the blocking buffer, Iba1 antibody (a microglial marker diluted 1:500 with the blocking buffer) was added and incubated overnight at 4°C. The sections were then washed three times with PBS for 5 min each. The secondary antibody (Alexa Fluor488 goat anti-rabbit, 1:500, protected from light) was diluted with blocking buffer and incubated at room temperature for 60 min in the dark. The secondary antibody was discarded, and the sections were washed three times with PBS for 5 min each at room temperature in the dark. The surface liquid was blotted dry, and a DAPI-containing anti-fluorescence quenching mounting medium was added. The sections were then covered with a coverslip and mounted with a mounting medium. The sections were then observed and photographed under a confocal microscope. ImageJ was used to quantify the Iba-1 immunofluorescence positive areas in each field of view.

[0142] like Figure 8 As shown, liver-specific knockout of Sdhaf4 activated retinal microglia, resulting in an increased number of migrating cells that migrated from the inner interstitial layer (IPL) to the inner nuclear layer (INL). Furthermore, the area of ​​Iba-1-labeled positivity significantly increased. These results indicate the production of animals with typical retinopathy.

[0143] 2. Analysis of retinal inflammatory factor levels

[0144] Analyze retinal inflammatory factors, including IL-1β, IL6, IL18, TNF-α, and HIF1α (primers used for analysis are shown in Table 2).

[0145] like Figure 9 As shown, liver-specific knockout of Sdhaf4 increased the levels of multiple retinal inflammatory factors, particularly IL-1β, IL6, IL8, IL18, TNF-α, and HIF1α.

[0146] Protein level analysis showed that the protein expression of both IL-1β and CD11b, a molecular marker of microglia, was significantly increased.

[0147] The above results indicate that animals exhibiting typical retinal lesions were obtained.

[0148] 3. Visual function testing – Electroretinography

[0149] Electroretinography (ERG) is a visual electrophysiological test used to assess visual function. Upon light stimulation, a small negative wave (negative in the cornea), called the a wave, initially appears, followed by a positive wave (positive in the cornea), called the b wave. The a wave primarily originates from the receptor potentials of photoreceptor cells; the b wave has a larger amplitude and is mainly related to the activity of bipolar cells.

[0150] Mice were dark-acclimatized for 24 hours beforehand, and the experiment was conducted in a dark room the following day. Compound tropicamide was instilled into the mice's eyes to induce mydriasis, and after approximately 5 minutes, sufficient mydriasis was confirmed using a red light flashlight. Mice were weighed and anesthetized with 140 μl of afodin per 10g injection, and oxbucaine was instilled into the eyes for surface anesthesia. Compound tropicamide eye drops were used to dilate the pupils, and the mice were placed on the experimental table. A 3 mm platinum wire loop electrode was placed on the corneal surface of the eye to record electrical signals. A subcutaneous needle electrode inserted into the base of the right leg served as ground, and another electrode placed under the skin of the head served as a common reference electrode. A / b waves were recorded and statistically analyzed using stimulation intensities (0.01, 0.1, 1, 3, 10 cd·s / m²).

[0151] like Figure 10 As shown, liver-specific knockout of Sdhaf4 significantly reduced b-waves and decreased visual function in mice at wavelengths of 3 and 10 cd·s / m2, indicating typical retinal lesions in the animals.

[0152] Example 6: Drug Screening

[0153] The liver-specific knockout Sdhaf4 mice established in Example 4 were used as the test animal model.

[0154] The candidate substance was administered to the test mouse model to observe whether it had a therapeutic effect on mouse retinopathy. Simultaneously, knockout Sdhaf4 mice that were not given the candidate substance served as a disease control.

[0155] Analysis indicator a: Analyze retinal inflammatory factors IL-1β, IL6, IL8, IL18, TNF-α, and HIF1α. If the level of one or more of these inflammatory factors is significantly reduced compared to the control group mice, it indicates that the candidate substance is a candidate substance that can alleviate retinopathy in mice.

[0156] Analysis indicator b: Analyze the protein expression of CD11b, a molecular marker of microglia. If the protein expression of CD11b is significantly reduced compared to the control group, it indicates that the candidate substance has a mitigating effect on mouse retinopathy.

[0157] Analysis index c: Visual function was analyzed by electroretinography. If the b wave was significantly improved compared with the control group mice at wavelengths of 3 and 10 cd·s / m2, and visual function was enhanced, it would indicate that the candidate substance was a candidate substance that could alleviate retinal lesions in mice.

[0158] Analysis indicator d: Morphological analysis of retinal microglia was performed (as described in Example 5), and the Iba-1 immunofluorescence positive area in each field of view was quantified using ImageJ. If the positive area of ​​Iba-1 labeling was significantly reduced compared with the control group mice, it indicated that the candidate substance was a candidate substance with alleviating effect on mouse retinopathy.

[0159] After performing the analysis of indicators 1, 2, 3 or 4 above, candidate substances that alleviate retinopathy in mice were collected.

[0160] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. A method for preparing animals with retinopathy, comprising downregulating the Sdhaf4 gene in the liver of the animal.

2. The method of claim 1, wherein, The downregulation targets the region of exons 1, 2 and / or 3 of the liver Sdhaf4 gene.

3. The method of claim 1, wherein, The animal is a rodent; preferably, it includes mice, rats, hamsters, and rabbits; more preferably, it is a mouse; and preferably, the retinopathy animal is an animal with normal blood sugar levels but retinopathy.

4. The method as described in claim 1 or 2, characterized in that, The downregulation includes: specific knockout of the liver Sdhaf4 gene using Cre-loxP-mediated recombination; Preferably, the loxP strategy is used to target the Sdhaf4 locus, with a pair of loxP sites flanking exons 1, 2 and / or 3, so that the exons are deleted after Cre-loxP-mediated recombination; More preferably, animals with a pair of loxP sites flanking exons 1, 2 and / or 3 of the Sdhaf4 locus in their genome are crossed with Alb-Cre animals to specifically knock out the liver Sdhaf4 gene.

5. The method as described in claim 1 or 2, characterized in that, The downregulation includes: infecting animals with a virus carrying a Sdhaf4 interfering molecule; preferably, the Sdhaf4 interfering molecule is shRNA; more preferably, the shRNA is shRNA with a sense strand CGUCUGGAGAGCAGCAAGATT and an antisense strand UCUUGCUGCUCUCCAGACGTT.

6. The method as described in claim 1 or 2, characterized in that, The animal models of retinopathy presented phenotypes selected from the following: Activation of retinal microglia increases the number of migrating cells, which migrate from the inner retinal layer to the inner nuclear layer; The positive area of ​​Iba-1 markers increased significantly; Increased levels of retinal inflammatory factors; preferably, the retinal inflammatory factors include: IL-1β, IL6, IL8, IL18, TNF-α, and HIF1α; Increased levels of CD11b protein, a molecular marker of microglia.

7. The application of the animal model of retinopathy prepared by the method of any one of claims 1-6, for: serving as an animal model for screening candidate drugs or therapeutic agents for the treatment of retinopathy; serving as an animal model for studying retinopathy; or for conducting drug metabolism and toxicology tests.

8. Application of reagents for downregulating the Sdhaf4 gene in the liver of animals in kits for preparing animal models of retinopathy; among which, The reagents are selected from: Sdhaf4 interfering molecule; preferably, the Sdhaf4 interfering molecule is shRNA; more preferably, the shRNA is shRNA with a sense strand CGUCUGGAGAGCAGCAAGATT and an antisense strand UCUUGCUGCUCUCCAGACGTT; or A reagent for specifically knocking out the liver Sdhaf4 gene; preferably, the reagent is a Cre-loxP-mediated recombination method for specifically knocking out the liver Sdhaf4 gene; more preferably, the reagent targets exons 1, 2 and / or 3 of the Sdhaf4 gene locus.

9. A method for screening candidate drugs or therapeutic agents for treating retinopathy, the method comprising: (1) Prepare an animal model of retinal disease using the method described in any one of claims 1-6 above; (2) The candidate substance was given to the animal model of (1) and the therapeutic effect of the candidate substance on retinal lesions was observed. If the symptoms of retinopathy are observed to improve in animal models, then the candidate substance is an animal substance for treating or alleviating retinopathy.

10. The method for screening candidate drugs or therapeutic agents for treating retinopathy as described in claim 9, characterized in that, observation middle: The transcriptional levels of retinal inflammatory factors IL-1β, IL6, IL8, IL18, TNF-α and / or HIF1α in the animal models were analyzed; if the levels of one or more inflammatory factors decreased significantly, the candidate substance was selected for treating or alleviating retinopathy in the animal. Analyze the protein expression of microglial cell molecular markers CD11b and IL-1β; if the protein expression of CD11b and IL-1β is significantly reduced, the candidate substance is a potential treatment for or to alleviate retinopathy in animals. If visual function is significantly enhanced by electroretinography (ERG) analysis, and b-waves are significantly increased, indicating improved visual function, then this candidate substance is suitable for treating or alleviating retinopathy in animals; and / or The analysis was performed by examining the Iba-1 immunofluorescence positive areas in the retina. If the positive area of ​​Iba-1 labeling was significantly reduced, the candidate substance was considered for treating or alleviating retinopathy in animals.

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