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Hematopoietic stem cell as well as preparation method and application thereof

A technology of hematopoietic stem cells and stem cells, applied in the field of hematopoietic stem cells, can solve problems such as inability to be treated clinically, have large side effects, and cannot be cured, and achieve the effects of reducing treatment risks, simple operation, and short time-consuming

Active Publication Date: 2018-06-01
广东赤萌医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] 1) Conservative treatment (regular long-term blood transfusion, iron removal therapy and splenectomy, etc.) Disadvantages: large side effects, high cost, no cure
However, the patient's autologous hematopoietic stem cells are genetically defective and cannot be directly used for clinical treatment.

Method used

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  • Hematopoietic stem cell as well as preparation method and application thereof
  • Hematopoietic stem cell as well as preparation method and application thereof
  • Hematopoietic stem cell as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Use the CRISPR-Sp Cas9 gene editing system to knock out the ZBTB7A gene, and the specific implementation steps are as follows:

[0050] 1. gRNA preparation

[0051] (1) according to the sequence design of ZBTB7A gene the gRNA sequence of 20nt, the target sequence of described gRNA is as shown in a kind of SEQ IDNO:1-SEQ ID NO:38;

[0052] (2) Synthesize the sense strand and antisense strand of the target sequence respectively (add cacc at the 5'-end of the sense strand, if the first nucleotide at the 5'-end of the sense strand is not guanine G, then add cacc at the 5'-end of the sense strand Add caccG at the -end; add aaac at the 5'-end of the antisense strand, if the first nucleotide at the 5'-end of the sense strand is not guanine G, then add C at the 3'-end of the antisense strand);

[0053] (3) The sense strand and antisense strand synthesized above were mixed, treated at 90°C, cooled naturally to room temperature for annealing treatment, and double-stranded DNA fr...

Embodiment 2

[0095] Use the CRISPR-Sa Cas9 gene editing system to knock out the ZBTB7A gene, and the specific implementation steps are as follows:

[0096] 1. gRNA preparation

[0097] (1) according to the sequence design of ZBTB7A gene the gRNA sequence of 21nt, the target sequence of described gRNA is as shown in one of SEQ IDNO:44-SEQ ID NO:38;

[0098] (2) Synthesize the sense strand and antisense strand of the target sequence respectively (add cacc at the 5'-end of the sense strand, if the first nucleotide at the 5'-end of the sense strand is not guanine G, then add cacc at the 5'-end of the sense strand Add caccG at the -end; add aaac at the 5'-end of the antisense strand, if the first nucleotide at the 5'-end of the sense strand is not guanine G, then add C at the 3'-end of the antisense strand);

[0099] (3) The sense strand and antisense strand synthesized above were mixed, treated at 90°C, cooled naturally to room temperature for annealing treatment, and double-stranded DNA frag...

Embodiment 3

[0140] The CRISPR-Sp Cas9 and CRISPR-Sa Cas9 gene editing systems were used respectively, combined with double gRNA to knock out the ZBTB7A gene. The specific implementation steps are as follows:

[0141] 1. The pX458 vector prepared in 3(6) of Example 1, which is connected with SEQ ID NO:26 and SEQ ID NO:37, is co-transformed into HEK 293T cells according to the method of 4 in Example 1, and then carried out according to the steps of 5 T7E1 enzyme digestion analysis of cutting efficiency;

[0142] 2. The pX601 vector prepared in 3(6) of Example 2, which is connected with SEQ ID NO:48 and SEQ ID NO:63, is co-transformed into HEK 293T cells according to the method of 4 in Example 2, and then carried out according to the step 5 T7E1 enzyme digestion analysis of cutting efficiency;

[0143] 3. Use 2% agarose gel electrophoresis to detect the effect of the above enzyme digestion, the results are as follows Figure 5 Shown: Each lane is named after the type of CRISPR system (Sp C...

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Abstract

The invention provides a hematopoietic stem cell. The hematopoietic stem cell is reduced in ZBTB7A gene expression amount or free from ZBTB7A gene expression amount. According to the invention, a CRISPR technique, when used for knocking out a fetal hemoglobin gene inhibiting factor, is simple and convenient to operate and short in time consumption; after the fetal hemoglobin gene inhibiting factor, namely ZBTB7A, is knocked out, influence to normal functions of nucleated red blood cells is avoided, and treatment means for thalassemia are widened; and by taking the autologous hematopoietic stemcell of the patient as a transplant, transplantation immunological rejection can be completely avoided and risks of stem cell treatment can be reduced.

Description

technical field [0001] The invention relates to a hematopoietic stem cell, in particular to a hematopoietic stem cell with no expression or decreased expression of ZBTB7A gene, its preparation method and application. Background technique [0002] The 2008 WHO Global Epidemiology Report on Hemoglobinopathies pointed out that about 71% of the population in 229 countries had hemoglobinopathies, a major health problem. Newborns born in these countries account for 89% of the world's newborns, of which more than 330,000 newborns per year are associated with hemoglobinopathy (83% of which are sickle cell disease and 17% are thalassemia). Hemoglobinopathies accounted for about 3.4% of deaths among children under 5 years of age. [0003] In my country, there are about 56,000 cases of β-thalassemia major each year, at least 30,000 of whom need standardized blood transfusions to survive, and about 5,500 cases of α-thalassemia major die of perinatal causes every year. Southern my coun...

Claims

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Application Information

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IPC IPC(8): C12N5/10A61K35/28A61P7/06
CPCA61K35/28C07K14/47C12N5/0647C12N2510/00
Inventor 祝海宝刘方方唐忆琳罗思施黄雨亭陶米林梁福才
Owner 广东赤萌医疗科技有限公司
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