HBB gene kit for correcting autologous hematopoietic stem cell of patient suffering from servious beta-thalassemia

A technology of hematopoietic stem cells and thalassemia, which is applied in blood/immune system cells, animal cells, gene therapy, etc., to achieve the effects of broad development prospects, high virus titers, and simple application and operation.

Inactive Publication Date: 2017-03-15
广东铱科基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the problem that there is no suitable gene reagent for treating β-thalassemia in the prior art, the purpose of the p

Method used

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  • HBB gene kit for correcting autologous hematopoietic stem cell of patient suffering from servious beta-thalassemia
  • HBB gene kit for correcting autologous hematopoietic stem cell of patient suffering from servious beta-thalassemia
  • HBB gene kit for correcting autologous hematopoietic stem cell of patient suffering from servious beta-thalassemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: Acquisition of specific HBB gene sequence

[0042] According to the human HBB gene sequence retrieved by NCBI, the retrieval sequence number is NC_000011, and specific primers were designed to obtain the full length of the HBB gene.

[0043] 1. Collect peripheral blood from normal people, and extract total DNA using the Chelex-100 method (see the instructions of the blood DNA extraction kit).

[0044] 2. Use Primer Premier 5 software to design specific primers as follows (SEQ ID No.1&SEQ ID No.2): HBB gene full-length clone upstream primer 5'-GGATCTTCCA GAGATTGTAC GGCTGTCATC ACTTAG-3' HBB gene full-length clone downstream primer 5'- CTGCCGTTCG ACGATTAAGG AACACTTCAGGGGAAAG-3'

[0045] 3. Carry out PCR amplification according to the reaction system in Table 1.

[0046] Table 1

[0047]

[0048] 4. Bio-Rad C1000 PCR instrument (Bio-Rad, USA) was used for PCR reaction. The reaction system was pre-denaturation at 98°C for 3min; Extend for 2 min, and end ...

Embodiment 2

[0052] Example 2: Construction of pSIN HBB-101 HIV lentiviral expression plasmid

[0053] 1. Use SpeI and NotI to digest the HBB-101 gene fragment, and digest at 37°C for 2 hours. The enzyme digestion reaction system is as follows:

[0054]

[0055] 2. Recover the pSIN HIV lentiviral expression plasmid by double digestion with SpeI and NotI and make it linear. Carry out the enzyme digestion reaction at 37°C for 2 hours according to the following table. The enzyme digestion reaction system is as follows:

[0056]

[0057] 3. Cloning Preparation

[0058] (1) connection

[0059] The reaction system is:

[0060]

[0061]

[0062] (2) conversion

[0063] ① Transfer 200 μl with a cooled sterile pipette tip to a sterile centrifuge tube, add 2 μl of connection solution, mix well, and place on ice for 30 minutes.

[0064] ②Heat in a constant temperature water bath at 42°C for 90 seconds, and then quickly place it on ice for 2 minutes.

[0065] ③ Add the ligated transfo...

Embodiment 3

[0074] Example 3: Construction of pSIN GFP HIV lentiviral expression plasmid

[0075] 1. Obtain the GFP gene sequence retrieved according to NCBI, the retrieval sequence number is NC_011521.1, and design specific primers to obtain the full length of the GFP gene.

[0076] 2. Use Primer Premier 5 software to design GFP gene-specific primers as follows (SEQ ID No.4&SEQ ID No.5):

[0077] GFP gene upstream primer: 5'-GGACTAGTTGAGTAAAGG-3'

[0078] GFP gene downstream primer: 5'-TTGCGGCCGCTTATTTGTA-3'

[0079] 3. Using the plasmid pEGFP-N3 as a template, the full-length GFP gene was cloned, and the GFP gene fragment was recovered by agarose gel electrophoresis.

[0080] 4. Use SpeI and NotI to digest the GFP gene fragment, and digest at 37°C for 2 hours. The enzyme digestion reaction system is as follows:

[0081]

[0082]

[0083] 5. Recover the pSIN HIV lentiviral expression plasmid by double digestion with SpeI and NotI and make it linear. Carry out the enzyme digestio...

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Abstract

The invention discloses an HBB gene kit for correcting autologous hematopoietic stem cells of a patient suffering from servious beta-thalassemia. The kit consists of a set of reagents for preparing a specific HBB-101 HIV slow virus and a set of reagents for HIV slow virus infection hematopoietic stem cells. The invention further discloses application of the kit in converting the autologous hematopoietic stem cells of the patient suffering from servious beta-thalassemia into hematopoietic stem cells with normal beta-globin synthesis functions. Experiment shows that the kit disclosed by the invention is simple to operate and relatively high in virus titer, and has wide development prospects in clinical study and treatment application, the virus infection efficiency is as high as 56.99%, and the purpose of treating the patient suffering from servious beta-thalassemia can be achieved through venous re-transfusion after PCR identification on corrected hematopoietic stem cells is implemented and the result of DNA sequencing identification is positive.

Description

technical field [0001] The invention relates to a gene therapy kit, in particular to an HBB gene kit for correcting autologous hematopoietic stem cells of severe beta-thalassemia patients. Background technique [0002] β-thalassemia is a hereditary hemoglobinopathy, which belongs to autosomal dominant inheritance. It is a disorder of β-globin synthesis caused by mutation of β-globin gene (HBB). It is the most common monogenic genetic disease in the world. [0003] The HBB gene is located in zone 1, zone 2, short arm of chromosome 11. Except for a few types of the disease, most of them are caused by point mutations (single nucleotide substitutions, additions or deletions). Mutations Those who cause partial inhibition of β chain synthesis are called "β + thalassemia"; those who cause complete inhibition of β chain are called "βo thalassemia". βo / βo homozygotes are severe, β+ / βo heterozygotes are mostly intermediate, and β+ / β is light. Due to the variety of β globin gene mutat...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10A61K48/00A61K35/28
CPCC12N15/86A61K35/28A61K48/0025A61K48/005C12N5/0647C12N2510/00C12N2740/15043
Inventor 邢晓王雯倩宋珂慧郭栋麦玉蝉陈莉郭伟
Owner 广东铱科基因科技有限公司
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