Method and product for repairing HBB gene of hematopoietic stem cell

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...

AI Technical Summary

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

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