Hematopoietic stem cells and erythrocytes capable of highly expressing gamma-globin, and preparation method and application of hematopoietic stem cells and erythrocytes
Through gene editing technology that inserts A base at specific locations of the HBG2 promoter, γ-globin expression is activated, and the problem of insufficient regulatory targets for γ-globin expression in β-thalassemia treatment is solved, achieving efficient and safe γ- Globin expression significantly improves the therapeutic effect of β-thalassemia.
Patent Information
- Application Number
- CN202310878578.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-18
- Publication Date
- 2025-01-17
AI Technical Summary
In the existing gene therapy methods for β-thalassemia, the γ-globin expression regulatory targets have not been fully utilized, which leads to the need to improve the safety and effectiveness of gene editing, and there is a risk of off-target effects.
By inserting a mutant of A base at positions -1368 and -1369 of the HBG2 promoter, gene editing is used to activate the expression of γ-globin, improve the fetal hemoglobin level, and prepare highly expressed γ-beads with sgRNA and Cas9 proteins. Protein hematopoietic stem cells and red blood cells.
It has achieved efficient activation of γ-globin expression in CD34+ hematopoietic stem cells. After 18 days of in vitro culture, the fetal hemoglobin level increased to 50%, reducing immune rejection, avoiding off-target effects caused by homology editing, and significantly improving treatment Safety and effectiveness of beta thalassemia.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of medicine, and particularly relates to a hematopoietic stem cell and red blood cell capable of highly expressing gamma-globin, and a preparation method and application thereof. Background Art
[0002] β-thalassemia (abbreviated as β-thalassemia) is a monogenic autosomal recessive inherited blood disease with the characteristics of disability and lethality. Its pathogenesis is due to the lack of expression of the β-globin peptide chain caused by a point mutation or large fragment deletion of the β-globin gene located on chromosome 11, which makes the α and β peptide chains unbalanced and unable to form hemoglobin with normal function, resulting in anemia in patients. At present, the treatment of β-thalassemia mainly relies on regular blood transfusions and bone marrow transplantation, which not only brings a heavy economic burden and great mental pressure to the patient's family, but also the matching and immune rejection of bone marrow transplantation and other problems also trouble many families. In recent years, with the rapid development of gene editing technology and gene therapy, β-thalassemia is also expected to be cured through gene therapy technology. Clinically, it has been found that fetal hemoglobin (α2γ2, a tetramer formed by α-globin encoded by the HBA gene and γ-globin encoded by the HBG gene, which is only expressed in the fetal period in normal people and slowly shuts down after birth) can alleviate the clinical symptoms of β-thalassemia patients if it is continuously expressed after birth. The explanation for this mechanism is that the continuously expressed γ-globin can compensate for the imbalance between α-like peptide chains and β-like peptide chains caused by β-globin deficiency. This discovery also provides a new entry point for the subsequent gene therapy of β-thalassemia, namely, reactivating the expression of the γ-globin gene.
[0003] At present, there are two gene therapy strategies for β-thalassemia. One is gene replacement therapy, which is to deliver the β-globin gene with normal function to autologous hematopoietic stem cells through lentivirus or adeno-associated virus vectors; the other is gene editing therapy, which is to repair the defective gene through gene editing technology, or edit the γ-globin modification gene, such as BCL11A and the quantitative trait locus located proximal to the HBG promoter (such as mutant hereditary persistent hemoglobin hyperplasia, HPFH), and deliver it to the patient's autologous hematopoietic stem cells through electroporation technology. There are also domestic biological companies that perform gene editing on BCL11A and HBG promoters. BCL11A has been proven to have the known function of γ-globin expression, and when the enhancer regulating BCL11A is defective, BCL11A is expressed and γ-globin expression is reopened. These studies have entered the clinical trial stage, but BCL11A has other important biological functions in addition to participating in the regulation of erythroid globin conversion, and the potential off-target effects brought about by gene editing may affect other life activities.
[0004] Therefore, there is still room for further exploration of editing targets that can more safely and effectively increase the expression of γ-globin. Summary of the invention
[0005] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a hematopoietic stem cell capable of highly expressing γ-globin.
[0006] Another object of the present invention is to provide a method for preparing the above-mentioned hematopoietic stem cells capable of highly expressing γ-globin.
[0007] One object of the present invention is to provide the above-mentioned hematopoietic stem cells capable of highly expressing γ-globin and the application of the preparation method thereof.
[0008] The purpose of the present invention is achieved through the following technical solutions:
[0009] A hematopoietic stem cell capable of highly expressing gamma-globin is a mutant HBG2 promoter having the following mutation: an A base is inserted between positions -1368 and -1369 of the wild-type HBG2 promoter.
[0010] The mutant HBG2 promoter has the following nucleic acid sequence: 5'-CC T A A ACACTTTGAGCAGATA.
[0011] The underlined T in the above sequence is the -1369th position of the wild-type HBG2 promoter, and the underlined A is the -1368th position of the wild-type HBG2 promoter. The base numbers on the promoter are arranged with the transcription start site (ie, TSS) as position 0.
[0012] The wild-type HBG2 promoter is the HBG2 promoter before artificial mutation.
[0013] The wild-type HBG2 promoter can be found in NC_000011.9 (5276012-5278011) (genome version number: hg19).
[0014] The hematopoietic stem cells capable of highly expressing γ-globin are heterozygotes.
[0015] The method for preparing hematopoietic stem cells capable of highly expressing γ-globin comprises the following steps: editing hematopoietic stem cells by homologous recombination or gene editing to obtain hematopoietic stem cells in which an A base is inserted between positions -1368 and -1369 of the wild-type HBG2 promoter, which are hematopoietic stem cells capable of highly expressing γ-globin.
[0016] The gene editing step is preferably as follows: transfecting the sgRNA and Cas9 protein whose nucleic acid sequences are shown below into primitive hematopoietic stem cells, culturing, and obtaining edited cells, i.e., hematopoietic stem cells that can highly express γ-globin;
[0017] sgRNA: 5'-tatctgctcaaagtgttagg-3'.
[0018] The sgRNA and the Cas9 protein are preferably mixed in a ratio of 300 pmol:18 μg.
[0019] The transfection method is preferably electrotransfection.
[0020] The electro-transfection procedure is preferably EO-100.
[0021] The primitive hematopoietic stem cells are preferably CD34 + cell.
[0022] The primitive hematopoietic stem cells are preferably derived from autologous β-thalassemia patients, thereby reducing immune rejection reactions.
[0023] The dosage of the primitive hematopoietic stem cells is preferably calculated based on 300 pmol sgRNA to 50,000 primitive hematopoietic stem cells.
[0024] The culture steps are preferably as follows: after transfection, the cells are allowed to stand, then washed with PBS, centrifuged, resuspended with culture medium, and then recovered at 37° C. and 5% CO 2 .
[0025] The standing time is preferably 10 minutes.
[0026] The centrifugation condition is preferably 300g centrifugation for 10 min.
[0027] The composition of the culture medium is preferably as follows: X-Vivo 15 serum-free culture medium, 100 ng / mL SCF, 100 ng / mL TPO (thrombopoietin), and 100 ng / ml Flt3-L.
[0028] The recovery culture time is preferably 24 hours.
[0029] The PBS used in the present invention is 0.01 mol / L PBS with a pH of 7.4.
[0030] After the hematopoietic stem cells that can highly express γ-globin differentiate into red blood cells, the expression of γ-globin (HBG) is reactivated, thereby increasing the level of fetal hemoglobin. In the in vitro culture and differentiation into red blood cells experiment, the level of fetal hemoglobin increased to about 50% after 18 days of in vitro culture. Therefore, the hematopoietic stem cells that can highly express γ-globin can be delivered to patients with β-thalassemia for treatment.
[0031] Therefore, the hematopoietic stem cells capable of highly expressing γ-globin are used in the preparation of drugs for treating β-thalassemia.
[0032] A red blood cell capable of highly expressing gamma-globin has a mutant HBG2 promoter with the following mutation: an A base is inserted between positions -1368 and -1369 of the wild-type HBG2 promoter.
[0033] The method for preparing the above-mentioned erythrocytes capable of highly expressing γ-globin comprises the following steps: amplifying and differentiating the above-mentioned hematopoietic stem cells capable of highly expressing γ-globin to obtain erythrocytes capable of highly expressing γ-globin.
[0034] The specific steps of the expansion and differentiation culture are preferably as follows: culture with EDM-1 medium from day 1 to day 7, culture with EDM-2 medium from day 8 to day 11, and culture with EDM medium after day 12;
[0035] The composition of EDM medium is: IMDM medium + 330μg / mL holo human transferrin + 10μg / mL insulin + 2U / mL heparin + 5% plasma AB + 3U / mL erythropoietin (EPO) + 1% L-glutamine + 1% double antibody; the unit of % is mass percentage; Plasma AB refers to the plasma extracted from the blood of people with AB blood type;
[0036] The composition of EDM1 medium is: EDM+10 -6 mol / L hydrocortisone + 100ng / mL SCF + 5ng / mL IL-3;
[0037] The composition of EDM-2 medium is: EDM + 100 ng / mL SCF.
[0038] The use of the above-mentioned red blood cells capable of highly expressing γ-globin in the preparation of drugs for treating β-thalassemia.
[0039] A gene editing method for activating γ-globin expression comprises the following steps:
[0040] (1) transfecting the sgRNA and Cas9 protein whose nucleic acid sequences are shown below into primitive hematopoietic stem cells to obtain edited cells;
[0041] (2) the edited cells are expanded and differentiated to obtain red blood cells expressing activated γ-globin;
[0042] sgRNA: 5'-tatctgctcaaagtgttagg-3'.
[0043] The sgRNA and the Cas9 protein described in step (1) are preferably mixed in a ratio of 300 pmol:18 μg.
[0044] The transfection method described in step (1) is preferably electro-transfection.
[0045] The electro-transfection procedure is preferably EO-100.
[0046] The amount of primitive hematopoietic stem cells used in step (1) is preferably calculated based on 300 pmol sgRNA to 50,000 primitive hematopoietic stem cells.
[0047] The culturing procedure described in step (1) is preferably as follows: after transfection, the cells are allowed to stand, then washed with PBS, centrifuged, resuspended with culture medium, and then recovered at 37°C and 5% CO2.
[0048] The standing time is preferably 10 minutes.
[0049] The centrifugation condition is preferably 300g centrifugation for 10 min.
[0050] The composition of the culture medium is preferably as follows: X-Vivo 15 serum-free culture medium, 100 ng / mL SCF, 100 ng / mL TPO (thrombopoietin), and 100 ng / ml Flt3-L.
[0051] The recovery culture time is preferably 24 hours.
[0052] The specific steps of the expansion and differentiation culture described in step (2) are preferably as follows: culturing with EDM-1 medium from day 1 to day 7, culturing with EDM-2 medium from day 8 to day 11, and culturing with EDM medium after day 12;
[0053] The composition of EDM medium is: IMDM medium + 330μg / mL holo human transferrin + 10μg / mL insulin + 2U / mL heparin + 5% plasma AB + 3U / mL erythropoietin (EPO) + 1% L-glutamine + 1% double antibody; the unit of % is mass percentage; Plasma AB refers to the plasma extracted from the blood of people with AB blood type;
[0054] The composition of EDM1 medium is: EDM+10 -6 mol / L hydrocortisone + 100ng / mL SCF + 5ng / mL IL-3;
[0055] The composition of EDM-2 medium is: EDM + 100 ng / mL SCF.
[0056] The targeting efficiency of the above gene editing method reaches 95%, and the majority of the mutations obtained are in the form of an A base inserted between the -1368 and -1369 positions of the HBG2 promoter. Therefore, this method is suitable for the preparation of hematopoietic stem cells that efficiently express γ-globin or red blood cells that efficiently express γ-globin.
[0057] Compared with the prior art, the present invention has the following advantages and effects:
[0058] (1) At present, gene editing of HBG promoter is based on the discovered natural mutation sites and BCL11A binding sites. The editing site is usually within -200bp of HBG promoter, that is, in the HBG proximal promoter. The mutant HPFH site located in the HBG proximal promoter will make gene editing extremely difficult and its safety remains to be tested because the two genes HBG1 and HBG2 are highly homologous. The editing target provided by the present invention is located at the distal end of the HBG promoter, there is no homology, and the off-target effect is effectively avoided. It also has the following beneficial effects: the target is a non-coding region and does not affect the function of the coding gene; the target is easy to edit and is not suitable for CD34 + In hematopoietic stem cells, the efficiency of electroporation editing can reach about 95%.
[0059] (2) The target provided by the present invention has a strong regulatory ability. After the hematopoietic stem cells obtained after editing the target are cultured in vitro for 18 days, the fetal hemoglobin level increases to about 50%.
[0060] (3) It has been shown to be effective against β 0 / β 0 and β 0 / β +CD34+ hematopoietic stem cells from thalassemia patients have a good ability to activate γ-globin gene expression. BRIEF DESCRIPTION OF THE DRAWINGS
[0061] Figure 1 This is a schematic diagram of the location of the editing target site located at the distal end of the HBG2 promoter.
[0062] Figure 2 This is a graph showing the editing efficiency of the editing target located distal to the HBG2 promoter in hematopoietic stem cells.
[0063] Figure 3 This is the result diagram of off-target situation.
[0064] Figure 4 The following are the qPCR, HPLC and flow cytometry results of γ-globin after editing the target site.
[0065] Figure 5 The edited CD34 + Figure 1. Monoclonal heterozygous sequencing results.
[0066] Figure 6 CD34 + Figure 1. qPCR results of monoclonal heterozygous γ-globin. DETAILED DESCRIPTION
[0067] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.
[0068] Example 1
[0069] A safe and effective method to activate γ-globin expression by targeting CD34 + The distal position of the γ gene promoter (i.e., HBG promoter) of hematopoietic stem cells is gene-edited, and the cells are cultured in vitro to differentiate into red blood cells, activating the expression of γ-globin and increasing the level of fetal hemoglobin.
[0070] Distal position of the γ gene promoter (genome version: hg19): chr11:5277363-5277383.
[0071] The distal position sequence of the γ gene promoter: tatctgctcaaagtgttagg.
[0072] The specific steps are as follows:
[0073] (1) Cell culture
[0074] The mobilized β 0 / β +Human CD34 + Hematopoietic stem cells, the sorted cells were cultured in vitro: the culture system on day 0 was: X-Vivo 15 serum-free medium + 100ng / mL SCF + 100ng / mL TPO (thrombopoietin) + 100ng / ml Flt3-L; after 24 hours of culture, electroporation was performed, and after electroporation, culture was continued for 24 hours using the culture system on day 0, and then entered into expansion and differentiation culture: that is, cultured with EDM-1 medium from day 1 to day 7, cultured with EDM-2 medium from day 8 to day 11, and cultured with EDM medium from day 12 to day 18 to obtain hematopoietic stem cells.
[0075] The composition of EDM medium is: IMDM medium + 330μg / mL holo human transferrin + 10μg / mL insulin + 2U / mL heparin + 5% plasma AB + 3U / mL erythropoietin (EPO) + 1% L-glutamine + 1% double antibody. The unit of % is mass percentage. Plasma AB refers to the plasma extracted from the blood of people with AB blood type.
[0076] The composition of EDM1 medium is: EDM+10 -6 mol / L hydrocortisone + 100ng / mL SCF + 5ng / mL IL-3.
[0077] The composition of EDM-2 medium is: EDM + 100 ng / mL SCF.
[0078] (2) Design of sgRNA
[0079] The sgRNA was designed online using the website (CRISPOR (tefor.net)). In order to insert A between the third and fourth bases from the end of the sequence of chr11:5277363-5277383 at the distal end of the γ gene promoter (i.e., between -1368 and -1369), the cleavage site of the designed sgRNA must be located at the third base from the end. The sgRNA sequence is: 5'-tatctgctcaaagtgttagg-3', and the specific position is as follows Figure 1 shown.
[0080] (3) Electroporation
[0081] Using the lonza 4D electroporation system, 300 pmol sgRNA (the solvent is ultrapure water, 10 μL ultrapure water dilutes 3 nmol sgRNA to 300 pmol / μL) and 18 μg Cas9 protein (IDT, catalog number 1081059) were mixed and allowed to stand at room temperature for 15 minutes, and then added to 50,000 hematopoietic stem cells obtained in step (1) together with 20 μL P3 electroporation solution, mixed and transferred to the electroporation cup for electroporation, and the electroporation program was EO-100. After electroporation, the cells were allowed to stand at room temperature for 10 minutes, then washed once with PBS, centrifuged at 300 g for 5 minutes, and resuspended in a medium prepared with X-Vivo 15 serum-free medium + 100 ng / mL SCF + 100 ng / mL TPO (thrombopoietin) + 100 ng / ml Flt3-L, and placed in a cell incubator for 24 hours, and then amplified and differentiated as described in step (1).
[0082] (4) Evaluation of editing efficiency and off-target effects
[0083] After the cells were electroporated, they were cultured for 5 days, and some cells were taken out to extract DNA for evaluation of editing efficiency: primers were designed near the editing site for PCR, followed by Sanger sequencing. PCR primers are shown in Table 1 below:
[0084] Table 1
[0085]
[0086] The PCR amplification system and amplification conditions are shown in Table 2 below:
[0087] Table 2
[0088]
[0089] For off-target effect rating, we first predicted off-target sites through the website Cas-OFFinder, then selected the top 13 off-target sites for verification, designed primers for these off-target sites, performed second-generation sequencing after PCR, and analyzed the sequencing results using Cas-Analyzer. The off-target site primers and PCR amplification conditions are shown in Table 3 below:
[0090] Table 3
[0091]
[0092] Editing efficiency Figure 2 As shown, the off-target situation is Figure 3 shown. Figure 2 The results showed that the editing efficiency in hematopoietic stem cells reached 95%, of which 66% were A insertions at positions -1368 and -1369. Figure 3The results showed that there were no obvious off-target effects of editing in hematopoietic stem cells.
[0093] (5) Flow cytometry to detect hematopoietic stem cell differentiation
[0094] Nucleated red blood cells specifically express CD235a and CD71 surface antigen molecules, so we used FITC-coupled CD235a (eBioscience, 11-9987-82) and PE-cy7-coupled CD71 flow cytometry antibodies (eBioscience, 25-0719-42) to detect the differentiation of hematopoietic stem cells cultured 18 days after transfection, so as to evaluate whether editing would affect the differentiation and maturation of red blood cells. The specific operation is as follows: Collect about 1×10 5 Place cells in a 1.5 mL clean centrifuge tube, centrifuge at 300 g for 5 minutes, discard the supernatant, wash once with PBS, and discard the supernatant after centrifugation. Add 5 μL of FITC-CD235a antibody and PE-CD71 antibody respectively, incubate at 4°C in the dark for 30 minutes, then add 1 mL of PBS, centrifuge at 300 g for 5 minutes, discard the supernatant, wash twice with PBS, and finally resuspend the cells with 300 μL PBS for detection.
[0095] (6) qPCR, flow cytometry, and high-performance liquid chromatography (HPLC) were used to detect the expression level of γ-globin. Figure 4 shown. Figure 4 The results showed that editing in hematopoietic stem cells could significantly increase the expression level of γ-globin to about 40%.
[0096] (7) Screening of single clones: After the hematopoietic stem cell editing is completed, culture it for 24 hours, then use flow cytometry to sort the single clones and place them in a 96-well plate and continue to culture according to step (1). On the 14th day of culture, take out 1 / 10 of the cells to extract DNA, and then perform PCR amplification (the system and conditions are as shown in step (4)) and first-generation sequencing to perform genotyping. On the 18th day of culture, collect the cells to extract RNA for qPCR (primers, system and conditions are as shown in step (6)) to quantify the expression level of γ-globin. The genotyping results are as follows. Figure 5 shown. Figure 5 The results showed that the edited hematopoietic stem cells were heterozygous with A inserted between -1368 and -1369. Figure 6 shown. Figure 6 The results showed that the expression level of γ-globin in edited hematopoietic stem cell heterozygotes was significantly increased to about 50%.
[0097] qPCR:
[0098] RNA extraction: After 18 days of cell culture and differentiation, about 1×10 6Place cells in a 1.5mL clean centrifuge tube free of RNase, centrifuge at 300g for 5 minutes, discard the supernatant, wash once with PBS, and discard the supernatant after centrifugation. Add 1mL Trizol to break the cells. Add 200uL chloroform, mix well and let stand for 15 minutes, then centrifuge at 12000rpm in a 4℃ refrigerated centrifuge for 15 minutes, transfer the supernatant to a new clean 1.5mL centrifuge tube, add 500μL pre-cooled isopropanol, let stand for 10 minutes, then centrifuge at 4℃, centrifuge at 12000rpm for 10 minutes, discard the supernatant, add 1mL pre-cooled 75% ethanol, centrifuge at 8000rpm for 5 minutes, discard the supernatant, dry at room temperature, and add 50μL DEPC water to dissolve the precipitate.
[0099] Reverse transcription and qPCR: Total RNA was reverse transcribed into cDNA using PrimeScript RT reagent kit with gDNAEraser (Takara), and qPCR was performed using a two-step method using SYBR Premix Ex Taq II (Takara) on ABI Prism 7900 or BIO-RAD CFXmanager. The specific process is as follows:
[0100] Genomic DNA removal: Prepare the system according to Table 4 and react at 42°C for 2 minutes. Immediately transfer to ice after the reaction.
[0101] Reverse transcription of RNA into cDNA: Prepare the system according to Table 4 and react at 37°C for 15 minutes, then at 85°C for 5 seconds and immediately transfer to ice for storage.
[0102] qPCR reaction: After the qPCR reaction system is configured as shown in Table 5, the reaction is carried out using the conditions in Table 5. The qPCR primer sequences are shown in Table 6.
[0103] Table 4 Reverse transcription system
[0104]
[0105] Table 5 qPCR reaction system
[0106]
[0107] Table 6 qPCR primer sequences
[0108]
[0109] Flow cytometry detection of F cells:
[0110] CD34 + When the differentiation was cultured for 18 days, about 1×10 6Cells were washed with PBS and the supernatant was removed as much as possible. 4% paraformaldehyde was added to fix at room temperature for 20 minutes, and then 0.1% Triton-X 100 was added to break the membrane for 10 minutes. After washing with PBS, centrifugation was performed and the supernatant was discarded. 100 μL PBS was added to resuspend the cells, and FITC-coupled HbF flow cytometry antibody was added to incubate at room temperature for 30 minutes. Then, the cells were washed twice with PBS, and the cells were resuspended in 300 μL PBS and tested on the machine.
[0111] HPLC:
[0112] CD34 + When the differentiation was cultured for 18 days, about 1×10 7 After washing with PBS, remove the supernatant as much as possible, add 50-100μL ultrapure water to lyse the cells, and then perform three freeze-thaw cycles between dry ice and a 37°C water bath. The composition ratio of fetal hemoglobin (HbF) and adult hemoglobin (HbA) was analyzed using high-performance liquid chromatography Bio-Rad VARIANTβ-thalassemia Short Program.
[0113] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.
Claims
1. A hematopoietic stem cell capable of highly expressing γ-globin, characterized in that: The hematopoietic stem cell capable of highly expressing γ-globin has a mutant HBG2 promoter with the following mutation: an A base is inserted between the -1368th and -1369th positions of the wild-type HBG2 promoter.
2. The method for preparing hematopoietic stem cells capable of highly expressing γ-globin according to claim 1, characterized in that The method comprises the following steps: editing hematopoietic stem cells by homologous recombination or gene editing to obtain hematopoietic stem cells with an A base inserted between positions -1368 and -1369 of the wild-type HBG2 promoter, which are hematopoietic stem cells that highly express γ-globin.
3. The method for preparing hematopoietic stem cells capable of highly expressing γ-globin according to claim 2, characterized in that: The steps of gene editing are as follows: transfecting sgRNA and Cas9 protein with the nucleic acid sequence shown below into primitive hematopoietic stem cells, culturing, and obtaining edited cells, i.e., hematopoietic stem cells that highly express γ-globin; sgRNA: 5'-tatctgctcaaagtgttagg-3'.
4. The method for preparing hematopoietic stem cells capable of highly expressing γ-globin according to claim 3, characterized in that: The sgRNA and the Cas9 protein are mixed at a ratio of 300 pmol:18 μg; The transfection method is electrotransfection; The primitive hematopoietic stem cells are CD34 + cell; The amount of primitive hematopoietic stem cells is calculated based on 300 pmol sgRNA to 50,000 primitive hematopoietic stem cells; The culture steps are as follows: after transfection, the cells are allowed to stand, then washed with PBS, centrifuged, resuspended with culture medium, and then recovered and cultured at 37°C and 5% CO2.
5. An erythrocyte capable of highly expressing γ-globin, characterized in that: The erythrocytes highly expressing γ-globin have a mutant HBG2 promoter with the following mutation: an A base is inserted between positions -1368 and -1369 of the wild-type HBG2 promoter.
6. The method for preparing red blood cells capable of highly expressing γ-globin according to claim 5, characterized in that The method comprises the following steps: amplifying and differentiating the hematopoietic stem cells highly expressing γ-globin as claimed in claim 1 to obtain red blood cells highly expressing γ-globin.
7. Use of the hematopoietic stem cells capable of highly expressing γ-globin according to claim 1 or the red blood cells capable of highly expressing γ-globin according to claim 5 in the preparation of a drug for treating β-thalassemia.
8. A gene editing method for activating γ-globin expression, characterized in that The steps include: (1) transfecting the sgRNA and Cas9 protein whose nucleic acid sequences are shown below into primitive hematopoietic stem cells, culturing them, and obtaining edited cells; (2) The edited cells are then expanded and differentiated to obtain red blood cells that activate γ-globin expression; sgRNA: 5'-tatctgctcaaagtgttagg-3'.
9. The gene editing method for activating γ-globin expression according to claim 8, characterized in that: The sgRNA and the Cas9 protein described in step (1) are mixed at a ratio of 300 pmol:18 μg; The transfection method described in step (1) is electrotransfection; The amount of primitive hematopoietic stem cells in step (1) is calculated based on 300 pmol sgRNA to 50,000 primitive hematopoietic stem cells or primitive red blood cells; The steps of culturing in step (1) are as follows: standing after transfection, then washing with PBS, centrifuging, resuspending with culture medium, and recovering and culturing at 37° C. and 5% CO 2 ; The specific steps of the expansion and differentiation culture described in step (2) are as follows: culturing with EDM-1 medium from day 1 to day 7, culturing with EDM-2 medium from day 8 to day 11, and culturing with EDM medium after day 12; The composition of EDM medium is: IMDM medium + 330μg / mL whole human transferrin + 10μg / mL insulin + 2U / mL heparin + 5% plasma AB + 3U / mL erythropoietin + 1% L-glutamine + 1% double antibody; the unit of % is mass percentage; Plasma AB refers to the plasma extracted from the blood of people with AB blood type; The composition of EDM-1 medium is: EDM+10 -6 mol / L cortisol + 100ng / mL SCF + 5ng / mL IL-3; The composition of EDM-2 medium is: EDM + 100 ng / mL SCF.
10. Use of the gene editing method according to claim 9 in preparing hematopoietic stem cells that can efficiently express γ-globin or red blood cells that can efficiently express γ-globin.