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Kit for increasing fetal hemoglobin level in human red blood cells through genome base editing and applications thereof

A fetal hemoglobin, base editing technology, applied in extracellular fluid diseases, medical preparations containing active ingredients, blood diseases, etc., can solve problems such as hidden safety hazards, DNA double-strand breaks, complicated procedures, etc.

Pending Publication Date: 2019-07-23
国家卫生健康委科学技术研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the above methods have made significant breakthroughs, if these strategies are used clinically, there are certain limitations: ①Patients with β-thalassemia have different mutation sites in the β-globin gene, and about hundreds of mutations have been found so far For treatment with the above methods, different gene editing sites must be designed for each patient, resulting in high treatment costs and complicated procedures; ②iPSCs have a certain possibility of canceration, and there are still risks in clinical use; ③Technology for inducing iPSCs into HSPCs Immature; ④The use of CRISPR / Cas9 gene editing technology will produce DNA double-strand breaks, induce apoptosis, and cause safety hazards, such as the deletion of large fragments (Adikusuma et al., 2018), etc.
[0005] In order to improve the efficiency of base mutation and avoid the safety problems caused by DNA double-strand breaks, a method to obtain bases efficiently without causing double-strand breaks by fusing cytosine deaminase to dCas9 protein Tools for site-directed mutagenesis were developed

Method used

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  • Kit for increasing fetal hemoglobin level in human red blood cells through genome base editing and applications thereof
  • Kit for increasing fetal hemoglobin level in human red blood cells through genome base editing and applications thereof
  • Kit for increasing fetal hemoglobin level in human red blood cells through genome base editing and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] In this example, a base editing tool is used to combine sgRNA on a cell line, and this method will be implemented using a base editing tool (BE3, xBE3, ABE, BE-PLUS or BE-PLUS (AID)) and a plasmid form of the sgRNA ( figure 1 ).

[0066] 1.1 Plasmid construction

[0067] In the γ-globin (HBG) gene promoter -114bp, -117bp, -158bp, -175bp, -196bp, -198 / 199bp (take the transcription start site TSS as O point, 5' end as negative, 3' The sgRNA (SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3) was designed at the position of positive) to synthesize oligos.

[0068] 114sgRNA:

[0069] The upstream sequence is: 5'-ACCGcttgaccaatagccttgaca-3' (SEQ ID NO.(13))

[0070] The downstream sequence is: 5'-AAACtgtcaaggctattggtcaag-3' (SEQ ID NO.(14))

[0071] 117sgRNA:

[0072] The upstream sequence is: 5'-ACCGgctattggtcaaggcaaggc-3' (SEQ ID NO.(15))

[0073] The downstream sequence is: 5'-AAACgccttgccttgaccaatagc-3' (SEQ ID NO.(16))

[0074] 158sgRNA:

[0075] The upstream sequence is: 5...

Embodiment 2

[0114] In this example, in the hematopoietic stem cells (HSCs) isolated from normal people, the base editing system was used to realize base editing in the form of mRNA and sgRNA RNA, and the expression changes of HBG were detected.

[0115] 2.1 sgRNA plasmid construction

[0116] In the γ-globin (HBG) gene promoter -114bp, -117bp, -158bp, -175bp, -196bp, -198 / 199bp (take the transcription start site TSS as O point, 5' end as negative, 3' The sgRNA synthesis oligos was designed at the position where the end is positive).

[0117] 114sgRNA:

[0118] The upstream sequence is: 5'-TAGGcttgaccaatagccttgaca-3' (SEQ ID NO.(26))

[0119] The downstream sequence is: 5'-AAACtgtcaaggctattggtcaag-3' (SEQ ID NO.(27))

[0120] 117sgRNA:

[0121] The upstream sequence is: 5'-TAGGgctattggtcaaggcaaggc-3' (SEQ ID NO.(28))

[0122] The downstream sequence is: 5'-AAACgccttgccttgaccaatagc-3' (SEQ ID NO.(29))

[0123] 158sgRNA:

[0124] The upstream sequence is: 5'-TAGGccctggctaaactccaccca-3...

Embodiment 3

[0267] In this example, in hematopoietic stem cells (HSCs) isolated from patients, the protein of the base editing system and the RNA of sgRNA are used to form RNP to realize base editing, and the expression changes of HBG are detected.

[0268] 3.1 sgRNA plasmid construction and in vitro transcription

[0269] With embodiment 2.

[0270] 3.2 Acquisition of patient HSC

[0271] Hematopoietic stem cells (HSCs) can be harvested from the patient's bone marrow using well-known techniques.

[0272] 3.3 Isolation, culture and electroporation of patient HSC

[0273] With embodiment 2.

[0274] The isolated CD34+ hematopoietic stem cells were cultured in StemSpan SFEM medium at a concentration of 6×104 / ml, and 100 ul / ml of cytokines were added to the medium: StemSpanTM CD34+Expansion Supplement (10X) (STEMCELL 02691). The cells were cultured in a 37°C incubator with 5% CO2 and pre-stimulated for 24 hours. Incubate the corresponding base editing system protein with the correspondi...

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Abstract

The invention provides a kit for increasing fetal hemoglobin level in human red blood cells through genome base editing and applications thereof. The kit comprises a base editing system and sgRNA aiming at a gamma-globulin gene promoter site. The present invention utilizes a base editing technology to change the transcription factor binding site of gamma-globulin gene promoter region by C-T and / or A-G base mutation so as to raise the expression level of gamma-globulin and to provide an efficient and safe method and a kit for treating hemoglobinopathy.

Description

technical field [0001] The present invention relates to a kit and method for increasing the level of fetal hemoglobin (HbF) in human erythrocytes through genome base editing. Background technique [0002] Hemoglobinopathy includes a variety of anemias caused by inherited changes in the structure or expression of hemoglobin, including changes in the molecular structure of hemoglobin chains (eg, sickle cell anemia), and reduced or absent synthesis of one or more of these chains (eg, thalassemia) . Diseases associated with beta-globulin are often referred to as beta-hemoglobinopathies. For example, β-thalassemia is caused by a partial or complete defect in the expression of the β-globin gene resulting in a lack or absence of hemoglobin A (HbA); sickle cell disease (SCD) is caused by a mutation in the β-globin structural gene (HBB) caused by mutations. Current treatment for most patients remains supportive and aims to relieve symptoms and treat complications. The only cure i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85A61K35/28A61P7/06
CPCA61K35/28A61P7/06C12N15/85
Inventor 马旭江雯金孝华李广磊
Owner 国家卫生健康委科学技术研究所