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A method and product for repairing hbb gene of hematopoietic stem cells

A cell and gene technology, applied in the field of hematopoietic stem cell HBB gene repair, can solve the problems of semi-random, inability to achieve lifelong cure, decline in curative effect, etc.

Active Publication Date: 2022-01-04
EAST CHINA NORMAL UNIV +1
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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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  • A method and product for repairing hbb gene of hematopoietic stem cells
  • A method and product for repairing hbb gene of hematopoietic stem cells
  • A method and product for repairing hbb gene of hematopoietic stem cells

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Embodiment Construction

[0020] 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;

[0021] 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. The sequence shown in 1.

[0022] 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 s...

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Abstract

The invention discloses a method and product for hematopoietic stem cell HBB gene repair. The method utilizes CRISPR-Cas9 gene editing technology to target knockout of codon 41 / 42(-TCTT) in β-thalassemia (thalassemia) ) technology, by designing and synthesizing sgRNA that can recognize and guide Cas9 protein to the target sequence of the target gene, mix it with Cas9 protein and electrotransduce it into β-thalassaemia codon 41 / 42 (‑TCTT) hematopoietic stem cells, and introduce homologous The recombinant donor efficiently restores the normal coding function of the amino acid at the mutation site and restores the normal expression of the β-globin gene. The present invention utilizes the existing gene editing technology to edit transfusion-dependent β-thalassemia codon 41 / 42 (-TCTT), which has high repair efficiency, and the repaired patient's hematopoietic stem cells can rebuild the patient's blood system and treat thalassemia after autologous transplantation .

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 Patents(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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