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Hematopoietic stem cell gene modification method for targeting hemoglobin HBB mutant gene

A technology of hematopoietic stem cells and hemoglobin, applied in the field of gene modification of hematopoietic stem cells, can solve the problems of expensive medical expenses

Active Publication Date: 2018-03-02
YINFENG BIOLOGICAL GRP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently there is no effective treatment for thalassemia, and the medical costs are high, patients and families are under enormous economic pressure and mental pain

Method used

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  • Hematopoietic stem cell gene modification method for targeting hemoglobin HBB mutant gene
  • Hematopoietic stem cell gene modification method for targeting hemoglobin HBB mutant gene
  • Hematopoietic stem cell gene modification method for targeting hemoglobin HBB mutant gene

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Experimental program
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Effect test

Embodiment 1

[0039] The design of embodiment 1 gRNA

[0040] Design gRNA based on CD41 / 42(-CTTT), the most common HBB mutation in Guangxi, Guangdong, China.

[0041] The normal sequence near the HBB gene exon2 is as follows (as shown in SEQ ID NO.5):

[0042] 5'-tcccaccctt agGCTGCTGG TGGTCTACCC TTGGACCCAG AGGTTCTTTG AGTCCTTTGG-3'.

[0043] The mutation sequence near the HBB gene exon2 is as follows (the italic part is the missing 4bp, CD41 / 42del CTTT type) (as shown in SEQ ID NO.6):

[0044]

[0045] The sequence of the gRNA designed to target the position near HBB exon2 is as follows (the sequence shown is the corresponding DNA sequence) (as shown in SEQ ID NO.7, 8) (using software to design, targeting at 250-500 bp upstream and downstream of the mutation position design):

[0046] gRNA1-20: 5'-GACCACCAGCAGCCUAAGGG-3';

[0047] gRNA2-21: 5'-GCCCAUAACAGCAUCAGGAG-3'.

[0048] Clone the gRNA sequence into the T7 promoter vector pcDNA3.1, transcribe the gRNA by T7 RNA polymerase in vi...

Embodiment

[0056] 100ml of umbilical cord blood or 50-100ml of bone marrow of patients with thalassemia, after mononuclear cells were separated by Ficoll, CD34+ cells were obtained with CD34 magnetic beads (Miltenyi CD34 MultiSort kit), and resuspended in StemSpan basal medium.

[0057] The HDR450bp sequence was cloned into rAAV (recombinant adeno-associated virus) to form rAAV-HDR450bp.

[0058] There are two types of experiments:

[0059] Scheme 1: spCas9-2.0 mRNA transcribed in vitro; gRNA synthesized in vitro; cloning and preparation of rAAV-HDR450bp (recombinant adeno-associated virus) ssDNA containing the repaired HBB CD41 / 42 delCTTT sequence. The above mRNA and gRNA electroporation (electroporation) into CD34+ cells (electroporation in 4mm cuvette, 550V / cm, 38ms). After culturing for 1 day, CD34+ cells were transfected with rAAV-HBB, and the cells were cultured in Stemspan medium (adding 100ng / ml SR1 1.0uM factor 5 of TOPIL6SCF Flt3), counting and adding culture medium every 2 da...

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Abstract

The invention discloses a hematopoietic stem cell gene modification method for targeting a hemoglobin HBB mutant gene. The method 1 comprises the following steps: electro-transforming gRNA1, gRNA2 andthe mRNA of spCas9-2.0 into CD34+ cells containing the hemoglobin HBB mutant gene; and culturing the cells for one day, and transferring rAAV-HBB into the CD34+ cells, and harvesting the cells. The method 2 comprises the following steps: mixing and recombining a Cas9 protein with the gRNA1 and the gRNA2, and electro-transforming the recombinant Cas9 protein into the CD34+ cells; and culturing thecells for one day, and transferring the rAAV-HBB into the CD34+ cells, and harvesting the cells. The CD41 / 42 (-CTTT) in the most common HBB mutation in Guangxi province and Guangdong province in China is targeted, no other mutation or insertion sequences are left in chromosomes after gene editing is completed, and the gene editing efficiency can reach 15-20%.

Description

technical field [0001] The invention relates to a hematopoietic stem cell gene modification method targeting hemoglobin HBB mutation gene, and belongs to the field of novel gene editing to correct genetic mutations. Background technique [0002] Thalassemia (Thalassemia) was first described by Cooley and Lee in 1925 as a severe anemia that occurred in Italian children. Since it was first discovered in the Mediterranean region, it was called thalassemia or thalassemia (referred to as thalassemia). The term review committee stipulated that it should be called "thalassemia anemia". Thalassemia is a chronic hereditary hemolytic anemia caused by the blockage or complete inhibition of the synthesis of one or more globin peptide chains, resulting in abnormal composition of hemoglobin components in the blood. Thalassemia is a common autosomal recessive genetic disease, the most common hereditary hemolytic anemia with the highest incidence rate in the world, and one of the most seri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N15/864C12N5/10
CPCC12N5/0647C12N9/22C12N15/86C12N15/907C12N2510/00C12N2750/14143
Inventor 黄昕华徐峰波庞勇刘倩李德柱唐正晓
Owner YINFENG BIOLOGICAL GRP
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