Application of ATG5 in the treatment of anemia and anemia-related diseases

Abstract

This invention belongs to the field of biomedical technology and discloses the application of ATG5 in the treatment of anemia and anemia-related diseases. This invention is the first to discover that inhibitors used to suppress the expression and / or function of the ATG5 gene or ATG5 protein in erythroid cells can promote erythropoiesis and increase hemoglobin levels. Therefore, inhibiting the expression and / or function of the ATG5 gene or ATG5 protein in erythroid cells can prevent, alleviate, and / or treat anemia. This invention also provides a method for constructing an erythroid cell conditionally specific ATG5 knockout animal model. This method uses gene editing technology and / or RNA interference technology to prevent or suppress the expression of the ATG5 gene in the erythroid cells of the target animal. The erythroid cell conditionally specific ATG5 knockout animal model constructed by this method can be used to study the pathogenesis of anemia or anemia-related diseases, the role of ATG5 in the hematopoietic mechanism of animal erythroid cells, and to screen candidate drugs for the treatment of anemia.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of ATG5 in the treatment of anemia and anemia-related diseases. Background Technology

[0002] Red blood cells are the primary transporters of oxygen in the human body, and their stable quantity and function are fundamental to maintaining normal metabolism and life activities. Chronic low red blood cell counts and low hemoglobin production, as core characteristics of anemia, are widely present in various serious diseases such as chronic kidney disease, tumor-related anemia, inflammatory anemia, and myelodysplastic syndrome. This condition not only leads to persistent fatigue, weakness, and a severe decline in quality of life, but also causes tissue hypoxia, further increasing cardiac workload, leading to multiple organ dysfunction, and significantly increasing hospitalization and mortality rates. Therefore, increasing red blood cell production and improving anemia is a major clinical problem that urgently needs to be addressed, affecting the health of hundreds of millions of patients worldwide.

[0003] The fundamental solution to this problem lies in deeply analyzing the intricate regulatory network of erythropoiesis (erythroid hematopoiesis) and discovering novel drug targets that can effectively increase red blood cell production. Erythropoiesis is a complex process precisely regulated by multiple stages and factors; dysregulation of any key link can lead to insufficient output. Currently, standard treatments for chronic hypoerythrocyte counts (such as erythropoietin (EPO) infusion and iron supplementation) have limitations, including inconsistent response rates, significant side effects (such as increased risk of thrombosis), high cost, and ineffectiveness for certain types of anemia. This highlights the singularity and limitations of current therapeutic targets, urgently requiring us to explore the endogenous regulatory mechanisms of erythropoiesis from a completely new perspective in order to discover new targets that surpass existing therapies.

[0004] Autophagy, as an important metabolic regulation and quality control system in cells, plays an unclear role in the entire process of erythrocyte differentiation and maturation, especially whether it can serve as a key node in regulating erythrocyte production, which remains an unexplored area. ATG5 is the core executive gene of the autophagy pathway and an ideal entry point for studying autophagy function. However, exploring the role of ATG5 in erythropoiesis faces significant technical bottlenecks: existing systemic ATG5 gene knockout mouse models cannot be used for research due to neonatal death (Kuma et al., Nature 2004), limiting the functional study of this gene in adult erythroid hematopoiesis. Furthermore, because ATG5 is widely expressed in various tissues and organs throughout the body, even if a surviving model can be obtained, any erythrocyte-related phenotypes observed through systemic drug administration or systemic gene interference cannot rule out the possibility that they originate from systemic metabolic abnormalities, malnutrition, or other indirect effects on other tissues. Therefore, it is difficult to definitively attribute the observed phenomena to the autonomous function of ATG5 in erythroid cells. This non-specific function makes it impossible to accurately define the function and application of ATG5 in erythroid cells. Although gene knockdown using erythroid cell lines is a common in vitro research method, the results cannot accurately simulate gene function in the complex physiological environment of living organisms (such as the bone marrow microenvironment, systemic hormonal signaling, and intercellular interactions). The reliability and physiological relevance of in vitro experimental results are often questioned, making it difficult to draw reliable conclusions that can be validated in vivo.

[0005] In summary, there has long been a lack of an effective tool in this field to specifically intervene in potential regulatory targets (such as ATG5) in erythroid cells within mammals to study their impact on erythrocyte production. This lack of a tool severely hinders our progress in translating new biological discoveries into innovative therapeutic strategies for chronic erythrocyte hypocytosis. Therefore, developing an in vivo model that can directly study ATG5 function in erythroid cells within a physiological context has become a pressing technical need in this field. Summary of the Invention

[0006] In view of the problems and shortcomings of the existing technology, the purpose of this invention is to provide the application of ATG5 in the treatment of anemia and anemia-related diseases.

[0007] To achieve the objectives of this invention, the technical solution adopted is as follows: The first aspect of this invention provides the use of an inhibitor for suppressing the expression and / or function of the ATG5 gene or ATG5 protein in erythroid cells in any of the following: (A1) Application in the preparation of products that promote erythropoiesis; (A2) Application in the preparation of products that increase hemoglobin levels; (A3) Use in the preparation of products for the prevention, relief and / or treatment of anemia and / or anemia-related diseases.

[0008] According to the above application, preferably, the inhibitor is one or more substances selected from those that inhibit ATG5 gene expression, silence ATG5 gene, knock out ATG5 gene, knock down ATG5 gene, reduce ATG5 protein content, reduce ATG5 protein activity, or inhibit ATG5 protein function. Inhibition of ATG5 gene expression, silencing ATG5 gene, knocking out ATG5 gene, and knocking down ATG5 gene can be achieved through gene mutation, gene silencing, gene knockout, gene editing, or gene knockdown techniques well known to those skilled in the art. For example, RNA interference (RNAi) technology can specifically eliminate or shut down the expression of a specific gene; gene editing tools can be CRISPR / Cas9 technology, zinc finger nucleases (ZFNs), or transcription activator-like effector nucleases (TALENs), etc., but are not limited to these.

[0009] Gene knockdown technology, which inactivates or silences gene expression at the post-transcriptional or translational level, is well known to those skilled in the art. Such gene knockdown techniques include, but are not limited to, RNA interference, Morpholino interference, antisense nucleic acids, ribozymes, or dominant-negative repressive mutations.

[0010] It is well known in the art to silence the ATG5 gene by inhibiting its expression using shRNA or siRNA expressed by viruses (such as lentiviruses and adeno-associated viruses).

[0011] According to the above applications, preferably, the inhibitor is one or more of nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, peptides, proteins, gene editing vectors, lentiviruses, or adeno-associated viruses.

[0012] According to the above applications, preferably, the nucleic acid molecules include shRNA, microRNA, siRNA and / or antisense oligonucleotides.

[0013] According to the above application, preferably, the antibody is an antibody against the ATG5 protein, and the lentivirus or adeno-associated virus is a recombinant lentivirus or recombinant adeno-associated virus expressing the siRNA.

[0014] The second aspect of this invention provides the use of the ATG5 gene or ATG5 protein in screening candidate drugs that promote erythropoiesis and / or increase hemoglobin levels.

[0015] According to the above application, preferably, the screening method is as follows: screening drugs or reagents to be screened with ATG5 in erythroid cells as the target, and using drugs or reagents that can inhibit the expression and / or function of ATG5 gene or ATG5 protein in erythroid cells as candidate drugs to promote erythropoiesis or / and increase hemoglobin levels.

[0016] The third aspect of this invention provides the use of the ATG5 gene or ATG5 protein in screening candidate drugs for the prevention, relief, and / or treatment of anemia and / or anemia-related diseases.

[0017] According to the above application, preferably, the screening method is as follows: screening drugs or reagents to be screened with ATG5 in erythroid cells as the target, and using drugs or reagents that can inhibit the expression and / or function of ATG5 gene or ATG5 protein in erythroid cells as candidate drugs for the prevention, relief and / or treatment of anemia or / and anemia-related diseases.

[0018] The fourth aspect of the present invention provides a method for constructing an erythroid cell condition-specific knockout ATG5 animal model, comprising preventing or inhibiting the expression of the ATG5 gene in the erythroid cells of the target animal.

[0019] According to the above construction method, preferably, the ATG5 gene in the erythroid cells of the target animal is not expressed or its expression is suppressed by gene editing technology and / or RNA interference technology.

[0020] According to the above construction method, preferably, the expression of exon 3 of the ATG5 gene in the erythroid cells of the target animal is suppressed or not expressed by gene editing technology and / or RNA interference technology.

[0021] According to the above construction method, preferably, the gene editing technology is at least one of CRISPR / Cas9 technology, ZFN technology, TALEN technology and Cre-loxp technology; the RNA interference technology is at least one of small interfering RNA and short hairpin RNA.

[0022] According to the above construction method, preferably, the target animal is a mouse.

[0023] According to the above construction method, preferably, the construction method includes the following steps: transferring ATG... loxp / loxp Homozygous mice were crossbred with red-type specific Cre mice to obtain the ATG5 genotype. loxp / loxp Gypa-eGFP Cre+ / - mice were used to obtain an erythroid cell conditionally specific ATG5 knockout animal model; wherein, the erythroid-specific Cre tool mouse was a Gypa-eGFP-iCre+ / - mouse; the ATG... loxp / loxpHomozygous mice were obtained by inserting loxP sites at both ends of the third exon of the ATG gene using the Cre-loxp technology. loxp / loxp Homozygous mice.

[0024] According to the above construction method, preferably, the Gypa-eGFP-iCre+ / - mouse is the Gypa-eGFP-iCre+ / - mouse described in the literature "Construction and Functional Identification of Erythroid-Specific Gypa-Cre Mouse Model" (Geng Jingyu. (2021). Construction and Functional Identification of Erythroid-Specific Gypa-Cre Mouse Model [Master's Thesis]. Zhengzhou University.), and the Gypa-eGFP-iCre+ / - mouse is constructed according to the construction method described in that literature.

[0025] According to the above construction method, preferably, the ATG loxp / loxp The homozygous mice were constructed according to the method described in the literature "Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice" (T. Hara, et al., Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice, Nature 441 (7095) (2006) 885e889.).

[0026] According to the above construction method, preferably, ATG is used. loxp / loxp Homozygous mice were crossbred with red-type specific Cre mice to obtain the ATG5 genotype. loxp / loxp The specific procedure for Gypa-eGFP Cre+ / - mice is as follows: First, male ATG5 mice are... loxp / loxp Homozygous mice were mated with female Gypa-eGFP-iCre+ / - mice to produce offspring; the offspring mice with the ATG5 genotype were then selected. loxp / - Female mice with Gypa-eGFP-Cre+ / - and male ATG5 loxp / loxp When homozygous mice are mated, the genotype ATG5 is obtained. loxp / loxp Progeny mice of Gypa-eGFP Cre+ / -.

[0027] The fifth aspect of this invention provides the application of the erythroid cell condition-specific ATG5 knockout animal model constructed using the construction method described in the fourth aspect above in any of the following: (B1) Application in studying the pathogenesis of anemia or anemia-related diseases and / or the signaling pathways that cause anemia; (B2) Application in studying the role of ATG5 in the hematopoietic mechanism of animal erythroid cells; (B3) Application in screening inhibitors for suppressing the expression and / or function of the ATG5 gene or ATG5 protein in erythroid cells; (B4) Use in screening candidate drugs for the prevention, relief and / or treatment of anemia and / or anemia-related diseases.

[0028] Compared with the prior art, the positive and beneficial effects achieved by the present invention are as follows: (1) Previously constructed ATG5 systemic gene knockout mice died in the neonatal period (within one day after birth). Therefore, existing research suggests that the ATG5 gene is a core component of the pathway necessary for cell survival, and its functional loss must be harmful or detrimental to erythropoiesis. Moreover, since ATG5 systemic gene knockout mice are lethal in the neonatal period and cannot be used for research, the function of the ATG5 gene in adult erythropoiesis remains blank. This invention adopts a conditional gene knockout strategy, by knocking out ATG5 systemic gene knockout mice, the ATG5 gene is knocked out in the neonatal period and cannot be used for research. loxp / loxp Homozygous mice were crossbred with red line-specific Cre tool mice (Gypa-EGFP-iCre+ / -) to successfully produce mice with the genotype ATG5. loxp / loxp Gypa-eGFP Cre+ / - mice, due to the strict restriction of ATG5 gene knockout to erythroid cells, yielded an ATG5 genotype. loxp / loxp Gypa-eGFP Cre+ / - mice can survive to adulthood, allowing researchers to observe the role of ATG5 in the entire erythroid hematopoietic process in adult animals in a complete physiological environment for the first time in a long-term, dynamic manner. Therefore, the method for constructing an erythroid cell condition-specific knockout ATG5 animal model in this invention overcomes the technical biases in the prior art. The constructed erythroid cell condition-specific knockout ATG5 animal model can not only be used to study the pathogenesis of anemia and anemia-related diseases, but also to study the role of ATG5 in erythroid hematopoiesis. Furthermore, it successfully extends the time window for ATG5 functional research from the embryonic period to the entire life cycle, filling a key technical gap.

[0029] (2) This invention, by specifically knocking out the ATG5 gene in mouse erythroid cells, found that after knocking out the ATG5 gene, the number of red blood cells, the amount of hemoglobin and the hematocrit of mice with conditionally knocked-out ATG5 in erythroid cells were significantly increased, and the number of erythroid precursor cells in the bone marrow increased. This shows that specifically inhibiting the function of ATG5 in erythroid cells is a new and effective way to increase red blood cell production. Therefore, using the ATG5 gene or ATG5 protein in erythroid cells as a target can not only screen candidate drugs for promoting red blood cell production and increasing hemoglobin levels, but also screen candidate drugs for preventing, alleviating and / or treating anemia and / or anemia-related diseases.

[0030] (3) The erythroid cell conditionally knocked-out ATG5 mouse model constructed in this invention exhibits a stable "high erythrocyte production" phenotype, perfectly mimicking the therapeutic effect of successfully inhibiting the ATG5 target in erythroid cells. Therefore, this model can be directly used as in vivo evidence of "successful target inhibition effectiveness," thereby screening small molecule compounds, biological agents, or nucleic acid drugs that can specifically inhibit ATG5 expression or function in erythroid cells as candidate drugs for the prevention, relief, and / or treatment of anemia and anemia-related diseases. Moreover, the selected candidate molecules, because their mechanism of action is consistent with the model phenotype and have been validated in principle in physiological systems, have a much higher success rate and predictive value for clinical translation than traditional in vitro screening. This greatly accelerates the process from target discovery to drug development. Attached Figure Description

[0031] Figure 1 This diagram illustrates the construction process of an erythroid-specific ATG5 knockout mouse model and the genotype identification results; where A is a schematic diagram of the construction process; and B is the genotype identification results. Figure 2 The band results of Western blot analysis of ATG5 knockout efficiency in erythroid cells; Figure 3 Quantitative statistical results of Western blotting of ATG5 knockout efficiency in erythroid cells; Figure 4 The results of routine blood analysis for a condition-specific knockout ATG5 mouse model of erythroid cells; Figure 5 This is a statistical result of the number of erythroid precursor cells in the bone marrow; Figure 6 The results show the proportion of bone marrow erythroid precursor cells in living cells. Detailed Implementation

[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0033] The following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments of the invention. Experimental methods in the following embodiments that do not specify specific conditions employ conventional techniques in this art or follow the conditions recommended by the manufacturer; reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0034] Example 1: Construction of a conditionally specific knockout ATG5 mouse model of erythroid cells Male ATG5 loxp / loxp Homozygous mice were mated with female erythroid-specific Cre mice (the erythroid-specific Cre mouse is the Gypa-eGFP-iCre+ / - mouse) to obtain offspring; the offspring mice with the ATG5 genotype were then selected. loxp / - Female Gypa-eGFP-Cre+ / - mice and male ATG5 mice loxp / loxp The homozygous mice were mated to obtain the ATG5 genotype. loxp / loxp The progeny mice of Gypa-eGFP Cre+ / - are the erythroid cell condition-specific knockout ATG5 mouse model (e.g. Figure 1 (As shown in A). The erythroid-specific Cre tool mouse is the Gypa-eGFP-iCre+ / - mouse, which is the Gypa-eGFP-iCre+ / - mouse described in the literature "Construction and Functional Identification of Erythroid-Specific Gypa-Cre Mouse Model" (Geng Jingyu. (2021). Construction and Functional Identification of Erythroid-Specific Gypa-Cre Mouse Model [Master's Thesis]. Zhengzhou University.). The Gypa-eGFP-iCre+ / - mouse was constructed according to the construction method described in that literature. The ATG5... loxp / loxp Homozygous mice were obtained by inserting loxP sites at both ends of the third exon of the ATG gene using the Cre-loxp technology. loxp / loxp Homozygous mice. Preferably, the ATG loxp / loxpHomozygous mice were constructed according to the method described in the literature "Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice" (T. Hara, et al., Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice, Nature 441 (7095) (2006) 885e889.). The specific construction method described in the literature is as follows: a 1 kb XbaI–SpeI genomic fragment containing the predicted third exon of the ATG5 gene was selected, loxP sites were inserted on both sides of it and a neoʳ resistance selection cassette derived from pMC1-Neo was linked in the middle, and the DT-A gene was inserted downstream of the short homologous arm to construct a targeting vector. The linearized vector was electroporated into CCE embryonic stem cells in a 129 / SvEv background. Positive ES cells were injected into C57BL / 6 blastocysts to obtain chimeric mice. Chimeric mice were mated with C57BL / 6 mice to obtain ATG5. flox / + Heterozygous mice. ATG5 flox / + Heterozygous mice were mated and screened for homozygous ATG5 offspring. flox / flox Mice.

[0035] The specific methods for genotypic PCR identification and screening of offspring mice obtained through mating and breeding are as follows: 1. Extraction of mouse genomic DNA On day 21 after birth, a 0.3-0.5 cm piece of mouse tail tissue was cut and placed in a sterile 1.5 mL centrifuge tube. 500 μL of mouse tail lysis buffer (preparation of mouse tail lysis buffer reagent is shown in Table 1) and 2 μL of proteinase K were added to the centrifuge tube. After thorough mixing, the tube was placed in a 55℃ water bath for overnight digestion. The next day, the centrifuge tube was gently inverted to confirm that the tissue had been fully lysed. The centrifuge tubes were then placed in a centrifuge and centrifuged at 14,000 rpm for 10 minutes. The supernatant was carefully transferred to new centrifuge tubes labeled with the corresponding ear tags, and the bottom precipitate (containing rat tail remnants and impurities) was discarded. An equal volume of isopropanol was added to the supernatant, and the tubes were gently inverted to mix until filamentous DNA precipitate was observed to clump together. The tubes were centrifuged at 14,000 rpm for 1 minute, the supernatant was discarded, and the DNA precipitate at the bottom of the tubes was retained. 500 μL of 75% ethanol was added to the precipitate, and the tubes were gently resuspended. The tubes were then centrifuged at 14,000 rpm for 1 minute at room temperature, and the supernatant was discarded. The centrifuge tubes were inverted on absorbent paper and allowed to air dry after the ethanol had evaporated. 100 μL of sterile ddH2O was added to each tube, and the precipitate was thoroughly resuspended to obtain the genomic DNA solution.

[0036]

[0037] 2. Genotyping primers and PCR reaction The extracted mouse genomic DNA was used as a template for PCR amplification for genotype identification experiments.

[0038] The primers, reaction procedures, and reaction systems used in the PCR amplification reaction for genotyping identification of Gypa-eGFP-iCre+ / - mice are shown in Tables 2, 3, and 4, respectively. The primers were synthesized by the company.

[0039]

[0040]

[0041]

[0042] The primers, reaction procedures, and reaction systems used in the PCR amplification reaction for ATG5-loxp mouse genotype identification are shown in Tables 5, 6, and 4, respectively. The primers were synthesized by the company.

[0043]

[0044]

[0045] After the PCR amplification reaction, the genotype identification results were detected by agarose gel electrophoresis of the PCR amplification products. The results are as follows. Figure 1 As shown in B, by Figure 1 As shown in B, the erythroid cell condition-specific knockout ATG5 mouse model was successfully constructed.

[0046] Example 2: Western blot analysis of ATG5 knockout efficiency in erythroid cells of a conditionally specific ATG5 knockout mouse model. 1. Experimental Method: Western blot analysis of ATG5 knockout efficiency in erythroid cells of a conditionally specific ATG5 knockout mouse model was performed using the following steps: (1) Bone marrow Ter119 + Cell enrichment: 1) Collection and counting: Collect individual cells from the bone marrow, count the cells using a hemocytometer, and record the total number of cells.

[0047] 2) Biotin incubation: For every 50 million cells, resuspend in 500 μL Buffer I, add 5 μL Ter119biotin, mix thoroughly, and incubate on ice for 30 minutes (resuspend the cells gently every 10 minutes during this period).

[0048] 3) Magnetic bead binding and washing: Add 180 μL of Buffer I buffer (prepared as shown in Table 7) and 50 μL of biotin beads to every 50 million cell suspension, mix thoroughly, and incubate at 4°C in the dark for 15 minutes. After incubation, wash the cells once with Buffer I.

[0049] 4) Magnetic sorting and washing: After filtering the cell suspension through a 70 μm filter, load it onto an LS sorting column equilibrated with Buffer I; wash with 1 mL of Buffer I 10 times, and after removing the column, elute the cells with 1 mL of Buffer I. Load the eluent onto an MS sorting column equilibrated with Buffer I; wash with 0.5 mL of Buffer I 3 times, and after removing the column, elute the cells with 1 mL of Buffer I.

[0050] 5) Collection and storage: Collect the eluted Ter119⁺ positive cells, count them again, and record the cell count. After cell precipitation, store the cells in an ultra-low temperature freezer at -80℃ for later use.

[0051]

[0052] (2) Protein Western Blot Detection Bone marrow Ter119 + Total protein extraction from cells: Collect cells, centrifuge at 300 g for 10 min, and discard the supernatant. Wash twice with PBS, centrifuge at 300 g for 10 min, discard the supernatant, and store cells at -80℃ or lyse directly. Prepare a lysis buffer containing RIPA, protease inhibitor, and phosphatase inhibitor according to the specified ratio, and lyse on ice for 30 min, vortexing once every 10 min. Centrifuge at 14000 g for 10 min at 4℃, and collect the supernatant as total protein.

[0053] BCA protein quantification: Dilute 3 μL of protein sample 10-fold with 27 μL of ddH2O, and load 20 μL onto the plate. Prepare 8 gradients of BSA standards (2~0.0125 mg / mL). Prepare BCA working solution at a ratio of 49:1, add 200 μL to each well, and incubate at 37℃ for 30 min. Measure the absorbance at 562 nm using a microplate reader, plot a standard curve, and calculate the protein concentration.

[0054] Protein denaturation: Take a quantitative protein sample, add 5× loading buffer to a final concentration of 1×, mix well, and then boil in a water bath for 5 min. After denaturation, aliquot the protein and store at -80℃.

[0055] SDS-PAGE gel preparation: Clean and leak-test the glass plate, and prepare the gel using the Yaxin one-step method kit. Take 2.7 mL each of the lower gel solution and buffer, add 60 μL of AP, mix well, and pour into the gel; then take 750 μL each of the upper gel solution and buffer, add 15 μL of AP, mix well, pour into the gel, and insert the comb. Let it stand at room temperature for 15 min to solidify, then remove the comb.

[0056] Western Blot Detection: Prepare 1× electrophoresis buffer, load samples, and incubate at 60 V for 90 min. Assemble the transfer membrane system using a sandwich structure, and transfer at 100 V on ice for 60 min. Block the PVDF membrane with 5% BSA at room temperature for 1 h. Incubate with primary antibody overnight at 4℃, wash 3 times with PBST for 10 min each time; incubate with secondary antibody at room temperature for 2 h, wash 3 times with PBST for 10 min each time. Prepare chemiluminescence solution for color development, expose and photograph using an imager, and analyze the relative protein expression level using ImageJ grayscale analysis.

[0057] 2. Experimental Results: Ter119 from bone marrow in a conditionally specific knockout ATG5 mouse model of erythroid cells + Western blot analysis results of ATG5 knockout efficiency in erythroid cells are as follows: Figure 2and Figure 3 As shown.

[0058] Depend on Figure 2 and Figure 3 It can be seen that in mouse bone marrow Ter119 + In erythroid cells, conditional knockout mediated by Gypa-eGFP-iCre+ / - significantly reduced the expression level of ATG5 protein, with stable and significant knockout efficiency.

[0059] Example 3: Peripheral blood routine analysis of a conditionally knockout ATG5 mouse model of erythroid cells 1. Experimental Method: (1) Collection of mouse blood samples: The mice were anesthetized with isoflurane using a closed anesthesia device in a fume hood. After the mice reached a deep anesthesia state, the subsequent operations were carried out. Take a 1 mL sterile syringe, pre-inhale 50 μL LEDTA anticoagulant, collect about 1 mL of blood through cardiac puncture, collect it into a 1.5 mL centrifuge tube, and repeatedly invert and mix to avoid blood clotting.

[0060] (2) Blood routine test: 1) Turn on the blood routine instrument and wait for the instrument to complete the self-test before proceeding with the operation; enter the mouse information (species, sample number) into the computer, select the dilution factor and then load the sample for testing; after the test is completed, rinse the instrument and save the experimental data simultaneously.

[0061] 2. Experimental Results: Blood routine analysis results of the erythroid cell condition-specific knockout ATG5 mouse model are as follows: Figure 4 As shown.

[0062] Depend on Figure 4 It was found that after erythroid cell-specific knockout of ATG5, the number of erythrocytes, hemoglobin concentration, and hematocrit in the peripheral blood of mice were significantly increased, indicating the presence of an erythrocytosis phenotype. However, the indicators reflecting erythrocyte size and hemoglobin content (MCH, MCV, RDW) did not change significantly, indicating that ATG5 deficiency mainly affected the number of erythrocytes produced, and had no effect on the morphology and hemoglobin content of individual erythrocytes.

[0063] Example 4: Statistical analysis of the number of bone marrow erythroid precursor cells and their proportion in living cells 1. Experimental Method: (1) Preparation of mouse bone marrow single-cell suspension: 1) Mice were euthanized after being anesthetized with isoflurane, weighed, and the data recorded; 2) The mouse body surface was disinfected by spraying with 75% alcohol, and the femur and tibia were aseptically separated and placed on ice in a culture dish containing Buffer I for later use; 3) Buffer I was drawn with a 1 mL syringe and the bone marrow cavity of the femur and tibia was repeatedly rinsed until the bone marrow cavity turned white and no cells remained. The cells were collected in a 15 mL centrifuge tube; 4) The bone marrow cell suspension was passed through a 70 μm cell sieve to prepare a single-cell suspension and the volume was adjusted to 20 mL; 5) The suspension was thoroughly mixed, diluted 20 times with Buffer I, and then diluted 2 times with 2× trypan blue before counting and recording the total number of cells in both femurs and tibias. The cells were placed on ice for later use.

[0064] (2) Detection of mouse bone marrow erythroid cells: 1) Take 1×10 6 1) Centrifuge bone marrow cells at 300 g for 10 min, discard the supernatant, and resuspend the cells in 50 μL of the solution; 2) Add anti-mouse Fc blocking antibody to a final concentration of 2.5 μg / mL and block at room temperature for 10 min; 3) Add erythroid-specific fluorescent antibody according to the system (antibody ratios are shown in Table 8); 4) Incubate on ice in the dark for 30 min, gently thawing the cells 1-2 times during this period; 5) Wash the cells with 1 mL of Buffer I, centrifuge at 300 g for 10 min, and discard the supernatant; 6) Resuspend the cells in 200 μL of Buffer I and transfer them to a flow cytometer, add 2 μL of 7AAD, incubate at room temperature for 5 min, and then analyze the proportion and number of mouse bone marrow erythroid precursor cells.

[0065]

[0066] 2. Experimental Results: The statistical results of the number of erythroid precursor cells are as follows: Figure 5 As shown. By Figure 5 It is known that ATG5 erythroid-specific knockout does not affect the total number of bone marrow cells, but significantly increases the number of bone marrow erythrocyte precursors, suggesting that ATG5 plays an important regulatory role in erythroid development and may be involved in the differentiation or survival regulation of erythroid precursor cells.

[0067] The results of the analysis of the proportion of bone marrow erythroid precursor cells in living cells are as follows: Figure 6 As shown. By Figure 6 It can be seen that ATG5 erythroid-specific knockout significantly increased the proportion of nucleated erythrocytes in mouse bone marrow among living cells, consistent with previously observed increases in absolute numbers.

[0068] Finally, it should be noted that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any other way. Any person skilled in the art may use the above technical content as inspiration to make changes or modifications. These are equivalent embodiments with variations. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the technical concept of the present invention still fall within the protection scope of the claims of the present invention.

Claims

1. The use of inhibitors for suppressing the expression and / or function of the ATG5 gene or ATG5 protein in erythroid cells in any of the following: (A1) Application in the preparation of products that promote erythropoiesis; (A2) Application in the preparation of products that increase hemoglobin levels; (A3) Use in the preparation of products for the prevention, relief and / or treatment of anemia and / or anemia-related diseases.

2. The application according to claim 1, characterized in that, The inhibitor is one or more of the following: nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, peptides, proteins, gene editing vectors, lentiviruses, or adeno-associated viruses; the nucleic acid molecules include shRNA, microRNA, siRNA, and / or antisense oligonucleotides.

3. Application of ATG5 gene or ATG5 protein in screening candidate drugs that promote erythropoiesis and / or increase hemoglobin levels.

4. Application of ATG5 gene or ATG5 protein in screening candidate drugs for the prevention, relief and / or treatment of anemia and / or anemia-related diseases.

5. A method for constructing an erythroid cell condition-specific knockout ATG5 animal model, characterized in that, This includes suppressing or eliminating the expression of the ATG5 gene in the erythroid cells of the target animal.

6. The construction method according to claim 5, characterized in that, Gene editing and / or RNA interference techniques are used to suppress or prevent the expression of the ATG5 gene in the erythroid cells of target animals.

7. The construction method according to claim 6, characterized in that, Gene editing and / or RNA interference techniques can be used to suppress or prevent the expression of exon 3 of the ATG5 gene in the erythroid cells of target animals.

8. The construction method according to claim 7, characterized in that, The gene editing technology is at least one of CRISPR / Cas9, ZFN, TALEN, and Cre-loxp technologies; the RNA interference technology is at least one of small interfering RNA and short hairpin RNA; and the target animal is a mouse.

9. The construction method according to claim 8, characterized in that, Includes the following steps: ATG loxp / loxp Homozygous mice were crossbred with red-type specific Cre mice to obtain the ATG5 genotype. loxp / loxp Gypa-eGFP Cre+ / - mice were used to obtain an erythroid cell conditionally specific knockout ATG5 animal model; wherein, the erythroid-specific Cre tool mouse was a Gypa-eGFP-iCre+ / - mouse; the ATG... loxp / loxp Homozygous mice were obtained by inserting loxP sites at both ends of the third exon of the ATG gene using the Cre-loxp technology. loxp / loxp Homozygous mice.

10. The application of the erythroid cell condition-specific knockout ATG5 animal model constructed using any one of the construction methods described in claims 5-9 in any of the following: (B1) Application in studying the pathogenesis of anemia or anemia-related diseases and / or the signaling pathways that cause anemia; (B2) Application in studying the role of ATG5 in the hematopoietic mechanism of animal erythroid cells; (B3) Application in screening inhibitors for suppressing the expression and / or function of the ATG5 gene or ATG5 protein in erythroid cells; (B4) Use in screening candidate drugs for the prevention, relief and / or treatment of anemia and / or anemia-related diseases.

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