Method and product for repairing HBB gene of hematopoietic stem cell

A cell and gene technology, applied in the field of hematopoietic stem cell HBB gene repair, can solve the problems of inability to achieve life-long cure, high requirements for equipment and technology, semi-random, etc.

Active Publication Date: 2021-05-04
EAST CHINA NORMAL UNIV +1
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  • Abstract
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  • Claims
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Problems solved by technology

Among them, the use of lentiviral vectors for gene therapy has shown great potential, but the semi-random vector integration method has cancer risk
At the same time, the expression elements in the lentivirus will be gradually silenced during the long-term homing and self-renewal process of hematopoietic stem cells, which will reduce the curative effect, that is, it may not be possible to achieve the purpose of lifelong cure
In addition, the high-concentration and high-quality lentivirus required in clinical practice has extremely high requirements on equipment and technology, so it is difficult to reduce the cost

Method used

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  • Method and product for repairing HBB gene of hematopoietic stem cell
  • Method and product for repairing HBB gene of hematopoietic stem cell
  • Method and product for repairing HBB gene of hematopoietic stem cell

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Embodiment Construction

[0021] In one aspect, the present invention provides a method for repairing HBB (beta-globin gene) codon frameshift mutations in cells, including the steps of introducing nuclease and sgRNA into cells and editing the HBB gene, and the sgRNA guides The nuclease cuts the HBB gene and forms a break site; the codon frameshift mutation is a frameshift mutation caused by the mutation of codon 41 / 42 (-TCTT); the sgRNA targets the targeting sequence of the HBB gene Including the upstream 1bp of the codon 41 / 42 (-TCTT) site;

[0022] Preferably, the targeting sequence of the sgRNA targeting the HBB gene comprises the sequence shown in any one of SEQ ID No.1-7; more preferably, the targeting sequence of the sgRNA targeting the HBB gene comprises SEQ ID No. 1 for the sequence shown.

[0023] The nuclease is selected from one or more of Cas9, Cas3, Cas8a, Cas8b, Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1, Cpf1; preferably, the nuclease is Cas9; more preferably, the Cas9 is ...

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Abstract

The invention discloses a method and product for repairing the HBB gene of a hematopoietic stem cell. According to the method, the technology that missed site codon 41/42(-TCTT) in beta-thalassemia (thalassemia) is subjected to targeted knockout by utilizing a CRISPR-Cas9 gene editing technology; sgRNA capable of identifying and guiding Cas9 protein to the target sequence of a target gene is designed and synthesized; and the sgRNA and Cas9 protein are mixed and electrically transfected into the hematopoietic stem cell of the beta-thalassemia codon 41/42 (-TCTT), meanwhile, a homologous recombination donor is introduced to efficiently repair the normal coding function of amino acid at the mutation site and recover the normal expression of the beta-thalassemia gene. The transfusion dependent type beta-thalassemia codon 41/42 (-TCTT) is edited by utilizing the existing gene editing technology which has high repairing efficiency, the repaired hematopoietic stem cells of the patient can reconstruct the blood system of the patient and treat thalassemia after autotransplantation.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method and product for hematopoietic stem cell HBB gene repair. Background technique [0002] In recent years, an acquired immune mechanism used to resist the invasion of foreign DNA fragments such as phages and plasmids in bacteria and archaea has been elucidated. The system consists of Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CAS) genes. The immune interference process of the CRISPR system mainly includes three stages: adaptation, expression, and interference. In the adaptation phase, the CRISPR system will integrate a short DNA fragment from a phage or plasmid between the leader sequence and the first repeat sequence, and each integration is accompanied by the duplication of the repeat sequence, thereby forming a new repeat-spacer unit. In the expression phase, the CRISPR locus is transcribed into a CRISPR RNA (c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N9/22C12N5/10C12N15/90A61K35/28A61P7/06
CPCC12N15/113C12N9/22C12N15/907C07K14/47A61K35/28A61P7/06C12N2310/20C12N5/10C12N15/90C12N15/85
Inventor 吴宇轩杨菲席在喜张亮李大力刘明耀
Owner EAST CHINA NORMAL UNIV
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