Application of CRISPR (clustered regularly interspaced short palindromic repeats) gene editing technology in treatment of thalassemia

Abstract

The invention provides application of a CRISPR (clustered regularly interspaced short palindromic repeats) gene editing technology in treatment of thalassemia. A CRISPR system in the application comprises at least one nuclease and at least one sgRNA, the sgRNA can target an inhibiting element of an HBG (gamma globin gene) promoter; the nuclease can cut the inhibiting element of the HBG promoter, the sgRNA comprises a targeting sequence targeting the inhibiting element of the HBG promoter, and the targeting sequence comprises a sequence as shown in SEQ ID No.1 or a complementary sequence as shown in SEQ ID No.1. Transferring a compound of the nuclease and the sgRNA into a cell by an electrotransformation method can knock out the inhibiting element on the HBG (gamma globin gene) promoter, and the relative content of HBG mRNA in cells is improved, and compared with a traditional lentiviral infection technology, exogenous DNA random integration caused by viruses is avoided, and the prospect of clinical application is improved.

Description

technical field [0001] This application relates to the field of gene editing technology, in particular to the application of CRISPR gene editing technology in the treatment of thalassemia. Background technique [0002] β-thalassemia is a blood genetic disease caused by mutations in the β-globin gene. Currently, there are more than 300 known pathogenic mutations in this gene. Human hemoglobin is a heterotetramer composed of α and β globins. When β-globin is mutated and unable to express or lack of function, it will cause excessive accumulation of α-globin in red blood cells, resulting in abnormal formation of red blood cells and premature apoptosis of their precursors. So patients with beta thalassemia must have constant blood transfusions to maintain the beta globin levels in the body so that red blood cells can form normally. [0003] Long-term blood transfusion will lead to excessive accumulation of iron in the body, resulting in iron poisoning, so patients must receive ...

AI Technical Summary

Problems solved by technology

[0007] The use of plasmids to deliver Cas9 / sgRNA is not yet clinically possible

First of all, the efficiency of the plasmid delivery system is very low, but the toxicity is relatively high. It is temporarily impossible to meet sufficient editing efficiency for later transplantation to achieve therapeutic purposes.

Using lentivirus to infect hematopoietic stem cells is a common way to deliver Cas9 / sgRNA, but it takes a long time to infect (more than 72 hours) to achieve a relatively high efficiency; Integrated carcinogenic risk, after a long period of in vitro infection, those long-term hematopoietic stem cells that really play a role in self-renewal in transplantation begin to differentiate, and their stemness is weakened, which has a great impact on the success rate of stem cell transplantation in the later stage; in addition, lentivirus Infection still cannot achieve editing efficiency above 80%

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