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Methods and compositions for activating gamma-globin gene expression

A technology of gene expression and composition, applied in the field of gene editing, can solve the problems of unclear long-term efficacy and safety, and achieve the effect of improving gene expression and HbF ratio

Active Publication Date: 2021-09-03
GUANGZHOU REFORGENE MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These approaches have been shown to activate HbF expression, but the long-term efficacy or / and safety of these approaches are unknown and further development of new approaches is required

Method used

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  • Methods and compositions for activating gamma-globin gene expression
  • Methods and compositions for activating gamma-globin gene expression
  • Methods and compositions for activating gamma-globin gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Screening of ssODN and CRISPR-Cas systems for introducing an activating GATA element into the γ-globin gene promoter

[0039] By analyzing the sequences (SEQ ID NO: 1 and SEQ ID NO: 2) about 1.4kb upstream of the transcription start sites of HBG1 and HBG2, the inventors found that the sequences of many segments satisfy the substitution of a few bases , deletion, insertion and other changes can form the sequence structure of NTG-N(7-8)-WGATAR or NAG-N(7-8)-WGATAR. By analyzing whether there is an NGG (PAM recognized by spCas9) sequence near the target mutation base in these segments, and whether the cutting point is in the middle of the target sequence to be edited, five candidate targets that are most likely to achieve efficient gene editing were finally obtained area( Figures 2A to 2E ), according to the distance from the transcription start site from near to far: No. 1 region, between -92 and -66 of HBG1 and HBG2 promoters ( Figure 2A ); the second regi...

Embodiment 2

[0059] Example 2: ssODN and sgRNA-spCas9 vector co-transfect 293T cells for gene editing

[0060] The above 8 kinds of sgRNA-spCas9 vectors were co-transfected with appropriate ssODN into 293T cells to test whether the addition of ssODN could induce the expected mutation type after gene editing (HDR repair occurred). According to the position of the sgRNA cutting point, 8 kinds of sgRNA-spCas9 vectors were combined according to the sgRNA+ssODN combination method shown in Table 6 to form 30 combinations respectively. Using Lipofectamine 2000 TM Reagent, add ssODN while transfecting the plasmid, and deliver the ssODN and the plasmid together into the cells. The transfected cells were cultured for 4 days, and the cells were collected to extract genomic DNA. Since the gene editing efficiency of HBG1 and HBG2 is almost positively correlated, and the gene editing efficiency of HBG2 is often slightly higher than that of HBG1 ( image 3 ), so only the gene editing efficiency of the...

Embodiment 3

[0064]Example 3: Co-transfer of ssODN and sgRNA-spCas9 vector into K562 cells by electroporation for gene editing

[0065] Using liposomes (such as Lipofectamine 2000 TM Reagents) The efficiency of transferring plasmids into suspension cells is relatively low, and electroporation is generally used when transfecting suspension cells. Take the combination of some sgRNA and ssODN in Example 2, and introduce it into K562 cells through the lonza-4D electroporation instrument, and use the K562 cell transfection program that comes with the instrument. The transfected cells were cultured for 4 days, and the cells were collected to extract genomic DNA, and the HBG2 promoter gene fragment was amplified by PCR for sequencing. The gene editing efficiency of the sequencing results was analyzed by synthego software, showing that the combination of electroporated sgRNA-1, sgRNA-2, sgRNA-5 and ssODN in K562 can achieve a higher editing efficiency ( Figure 5 ), slightly higher than that of ...

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Abstract

The present invention relates to a novel method for activating transcription of gamma-globin gene. The present invention adopts the single-stranded oligonucleotide (ssODN) containing GATA or its antisense complementary sequence TATC as guidance information, carries out gene editing in the gamma-globin gene regulatory region to form an enhancer element containing GATA, which can promote gamma-globin The protein gene is expressed in mature red blood cells. The hematopoietic stem cells after gene editing using the technology of the present invention have normal functions, and can significantly increase the expression of fetal hemoglobin after being differentiated into red blood cells, so they can be used for clinical treatment of β-thalassemia and sickle cell anemia.

Description

technical field [0001] The invention relates to the technical field of gene editing, and relates to a method for activating gamma-globin gene expression, a composition and an application thereof. In this method, single-stranded oligonucleotide (ssODN) containing GATA or its antisense complementary sequence TATC is used as guidance information, and gene editing is performed in the promoter region of γ-globin gene to form an erythroid enhancer element containing GATA, which can promote The gamma-globin gene is expressed in mature erythrocytes. Background technique [0002] Hemoglobin (Hb) is a special protein that carries and transports oxygen in red blood cells. Hemoglobin is composed of globin and heme. Hemoglobin in adults is mainly a tetramer (α2β2) composed of 2 α-globin and 2 β-globin, which becomes adult hemoglobin (HbA). Mutations in the β-globin gene (HBB) can cause β-hemoglobin disorders (also known as β-hemoglobinopathy), including sickle cell disease (Sickle Cel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/67C12N15/90C12N15/12C12N5/10A61K35/28A61P7/06
CPCC12N15/113C12N15/85C12N15/67C12N15/907C07K14/805C12N5/0647A61K35/28A61P7/06C12N2310/20C12N2800/107C12N2830/001C12N2510/00C12N15/63C12N9/22A61K38/42A61K48/0066C12N15/11C12N2310/315C12N2800/80
Inventor 梁峻彬古博徐辉
Owner GUANGZHOU REFORGENE MEDICINE CO LTD
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