阅读说明：本技术 一种激活红细胞中γ珠蛋白基因表达的方法 (Method for activating gamma globin gene expression in erythrocyte ) 是由 徐湘民 赵存友 包秀勤 王忠局 叶宇华 邵聪文 于 2020-06-11 设计创作，主要内容包括：本发明公开了一种激活红细胞中γ珠蛋白基因表达的方法,公开了转录因子ERF及其相关元件在提高红细胞中γ珠蛋白基因HBG表达中的应用。通过对造血干细胞中转录因子ERF基因启动子区域的胞嘧啶脱氧核糖核苷酸位点甲基化修饰、敲除HBG2上游ERF的结合位点或者敲除HBG1下游ERF的结合位点,能够提高由此干细胞分化而来的红细胞中HBG表达,提高HbF的含量,从而降低α与非α-珠蛋白链比例或改善α-与非α-珠蛋白链比例失衡状态。这些新的靶点并对其编辑,对于β-地中海贫血的诊断和深层次的分子机制研究具有意义。(The invention discloses a method for activating gamma globin gene expression in erythrocytes, and discloses application of a transcription factor ERF and related elements thereof in improving the expression of the gamma globin gene HBG in the erythrocytes. The expression of HBG in erythrocytes differentiated from stem cells can be improved and the content of HbF can be increased by carrying out methylation modification on cytosine deoxyribonucleotide sites of transcription factor ERF gene promoter regions in hematopoietic stem cells, knocking out binding sites of ERF at the upstream of HBG2 or knocking out binding sites of ERF at the downstream of HBG1, so that the proportion of alpha to non-alpha-globin chains is reduced or the proportion imbalance state of the alpha to non-alpha-globin chains is improved. The new targets and the editing thereof have significance for the diagnosis and deep molecular mechanism research of beta-thalassemia.)

1. The application of the transcription factor ERF and related elements thereof in improving the expression of the gamma globin gene HBG in erythrocytes.

2. The use of claim 1, wherein the transcription factor ERF and its related elements are selected from at least one of:

cytosine deoxyribonucleotide of transcription factor ERF gene promoter region in hemopoietic stem cell is positioned as follows: chr19:42760664, chr19:42760707, chr19:42760743, chr19:42760757, chr19:42760764, chr19: 42760789;

binding sites for ERF upstream of HBG 2;

binding sites for ERF downstream of HBG 1.

3. The use according to claim 1 or 2, wherein the methylation modification of cytosine deoxyribonucleotide of transcription factor ERF gene promoter region of hematopoietic stem cell is carried out;

and/or, the binding site of the ERF upstream of HBG2 is edited by genetic methods;

and/or, the binding sites for ERFs downstream of HBG1 were edited by genetic methods.

4. Use according to claim 3, characterized in that the binding site SEQ ID No.1 of the ERF upstream of HBG2 is knocked out by gene editing; the binding site SEQ ID NO.2 of the downstream ERF of HBG1 was knocked out by gene editing.

5. The use of claim 3, wherein the sgRNA for methylation modification is

ERF1：Cccctcgttgggattttgtg，

ERF2：atgaagatgattattattgc。

6. The use of claim 4, wherein the sgRNA for the binding site knock-out of the ERF upstream of HBG2 is UEBS KO: GATACAACCACCTGCTCCAA are provided.

7. The use of claim 4, wherein the sgRNA for the binding site knock-out of the ERF downstream of HBG1 is DEBS KO 1: GGAACAACCAGCGGCCCTCG the flow of the air in the air conditioner,

DEBS KO2：TGTGCCAAATTCTGAGGCTG。

8. the application of the sgRNA of any one of claims 5 to 7 to increase expression of a gammagglobin gene HBG in erythrocytes.

9. A composition for increasing the expression of the gammagglobin gene HBG in erythrocytes, comprising the transcription factor ERF according to any one of claims 1 or 2 and its related elements.

10. The composition as claimed in claim 9, further comprising: a pharmaceutically acceptable carrier and/or adjuvant; other active ingredients that increase the expression of the gamma globin gene HBG in erythrocytes.

Technical Field

The invention belongs to the technical field of life science and technology, and particularly relates to a method for activating gamma globin gene expression in erythrocytes.

Background

Beta-thalassemia (beta-thalassemia, short for beta-thalassemia) is one of the most common Mendelian genetic diseases in the world, and is characterized by genetic mutations that reduce (or lack) the synthesis of the beta-globin peptide chain that makes up hemoglobin, resulting in severe lethal hemolytic disease. Beta-thalassemia patients have diseases in the second year of life after birth, need lifelong transfusion therapy, and patients who do not receive transfusion and other therapies have a life expectancy of 5 years, which seriously affects the quality of life. One of the phenotypic characteristics of beta-thalassemia is the imbalance in the ratio of alpha-to non-alpha-globin chains (including beta-, gamma-, and-chains), and the major cause of beta-thalassemia is the decrease in beta-globin synthesis due to mutations in the beta-globin gene, which inevitably results in an excess of alpha-globin chains in Hb tetramer (HbA: alpha 2 beta 2) synthesis, i.e., an increased ratio of alpha-to non-alpha-globin chains, which is the basis for the injury of beta-thalassemia red blood cells and a series of erythroid pathological changes. In the prior art, scholars are working on treatment regimens that alleviate the beta-thalassemia condition by reducing the alpha to non-alpha-globin chain ratio or improving the out-of-balance condition of the alpha to non-alpha-globin chain ratio.

Since the 50's of the last century, although the present inventors have made extensive knowledge of the composition and expression of beta-globin chain synthesis in human Hb at three different stages of development (embryonic, fetal and adult), the gamma chain constituting HbF rapidly decreases during fetal birth (gamma-chain synthesis is off) and is replaced by rapid beta-chain synthesis in adult hemoglobin (HbA) (beta-chain synthesis is on). In recent years, through research on GWAS, genetic families and gene functions, a key gene BCL11A for regulating the 'switch' effect is firstly discovered, and then two important genes, MYB and KLF1, involved in the regulation of the metabolic pathway are gradually elucidated, and the three genes are rapidly applied to clinic as evaluation indexes for accurate diagnosis of beta-thalassemia. The novel target, particularly BCL11A, is a potential important application target for beta-thalassemia stem cell/gene therapy by means of DNA Editing and other technologies. In the prior art, the expression of the reactivated gamma globin is mainly realized by taking BCL11A, MYB and KLF1 as targets and modifying or editing the targets.

Therefore, the search for a new potentially important application target of beta-thalassemia stem cell/gene therapy becomes a problem to be solved urgently.

Disclosure of Invention

The invention aims to provide application of cytosine deoxyribonucleotide of transcription factor ERF gene promoter region in hematopoietic stem cells, HBG2 upstream or HBG1 downstream binding site.

Another object of the present invention is to provide a method for activating the expression of the gamma globin gene (HBG) in erythrocytes.

Drawings

FIG. 1 shows the constructed pCDH-U6-sgRNA-EF1 α -dCas9-MQ1-T2A-GFP vector.

FIG. 2 is a graph of the level of site-specific methylation and the expression of ERF in accordance with the present invention, wherein A is a graph of the methylation level and B is a histogram of the expression of HbF. Methylation: methylation; control is Control group; dCas9-MQ1+ sgRNA methylation group.

FIG. 3 shows the results of gene knockout verification, where A is the insertion deletion distribution rule of UEBS knockout in HUDEP-2. Black rectangular boxes show the position of sgrnas. The red rectangle shows PAM. Dashed lines show the cleavage points of the sgrnas. B is the sequencing map of HUDEP-2 after DEBS knock-out. The present invention uses two sgRNAs to locate the DEBS fragment. The black dashed line indicates the cleavage site. C is the indel distribution law of UEBS knockouts in CD34+ hematopoietic stem cells. D is the sequencing profile of CD34+ hematopoietic stem cells knocking out DEBS. WT is wild type; DEBS, downstream ERF binding site; UEBS the upstream ERF binding site.

FIG. 4 shows the flow cytometric results after cell induction differentiation, where A is HUDEP-2 cells and B is CD34+ cells. Ctrl is blank control; DEBS, downstream ERF binding site; UEBS upstream ERF binding site knockout; KO-knock out of gene.

FIG. 5 shows QPCR and HPLC results after knocking-out binding sites UEBS and DEBS, A is QPCR of HUDEP-2 cells; b is HPLC of HUDEP-2 cells; c is QPCR of CD34+ cells; d is the HPLC result of CD34+ cells. Ctrl is blank control; DEBS, downstream ERF binding site; UEBS, an upstream ERF binding site; KO-knock out of gene.

Detailed Description

The technical solution of the present invention is clearly and completely illustrated below with reference to the following examples, but is not limited thereto.