Globin gene dual-expression lentivirus vector and application thereof

A technology for expressing vectors and viral vectors, applied in the direction of viruses/bacteriophages, viruses, vectors, etc., to achieve the effects of avoiding homologous recombination, high-efficiency expression, and reducing homology

Active Publication Date: 2020-07-03
INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, objectively, the length of the carrier is sh...

Method used

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  • Globin gene dual-expression lentivirus vector and application thereof
  • Globin gene dual-expression lentivirus vector and application thereof
  • Globin gene dual-expression lentivirus vector and application thereof

Examples

Experimental program
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Embodiment 1

[0078] The design of embodiment 1HBB and HBG2 fusion gene

[0079] According to the amino acid sequence of the wild-type HBB protein and the translation usage frequency of codons in the human proteome, redesign the coding sequence of the HBB protein, optimize the secondary structure of the 5'UTR, and remove minor cis-elements such as 3-HS, HBB - the enhancer of intron No. 2, etc., to obtain the first HBB expression cassette as shown in SEQ ID NO: 2, comprising a total of 50bp 5'-UTR and 441bp ORF;

[0080] Taking the first HBB expression cassette as the comparison object, mutating the wild-type HBB gene base by base, reducing the homology with the first HBB expression cassette, and obtaining the second HBB expression cassette as shown in SEQ ID NO:3;

[0081] The HBG2 expression cassette was initially optimized according to the codon usage frequency to obtain the first HBG2 expression cassette as shown in SEQ ID NO:4;

[0082] Optimize the 147 amino acids encoded by the ORF o...

Embodiment 2

[0086] The preparation of embodiment 2 lentiviral vectors

[0087] Artificially synthesized 1stHBB-P2A-T2A-2ndHBB, 1stHBB-P2A-T2A-1stHBG2, 1stHBB-P2A-T2A-2ndHBG2 fusion genes, and cloned HS2, HS3 and HS4 as shown in SEQ ID NO:6-8 from the genome , the HBB promoter βpro shown in SEQ ID NO: 9, the HBB polyA terminator shown in SEQ ID NO: 10, assembled by restriction endonuclease ligation and homologous recombination to obtain a complete globin gene double expression module, wherein, The full length of 1stHBB-P2A-T2A-2ndHBG2 is about 3.5kb, which saves about 2kb load compared with BB305. It is reverse loaded into the lentiviral vector and constructed as follows: figure 2 The indicated HBB and HBG2 double expression lentiviral vectors.

Embodiment 3

[0088] Example 3 Preparation of recombinant lentivirus

[0089] In this example, 293T cells were used to prepare recombinant lentivirus, and when the 293T cells spread to 80-90% of the bottom of a 100mm culture dish, the lentivirus was packaged:

[0090] 2 hours before virus packaging, replace the medium with DMEM containing 1% fetal bovine serum, and add 6mL / 100mm culture dish;

[0091] Prepare the plasmid mixture shown in Table 1:

[0092] Table 1

[0093] Reagent dose globin expression plasmid 7μg psPAX2 7μg pVSVG-Rev 7μg

[0094] Expression plasmids include: lentiviral vectors expressing 1stHBB-P2A-T2A-2ndHBB, lentiviral vectors expressing 1stHBB-P2A-T2A-1stHBG2, or lentiviral vectors expressing 1stHBB-P2A-T2A-2ndHBG2. Add to 500μL opti-MEM medium, mix well;

[0095] Add 36 μg PEI to another 500 μL opti-MEM medium, mix well, and let stand at room temperature for 5 minutes;

[0096] Mix the plasmid with PEI, mix well by pipetting, a...

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Abstract

The present invention provides a globin gene dual-expression lentivirus vector and an application thereof. The lentiviral vector comprises optimized multiple globin genes connected by P2A-T2A connecting peptides. The efficient expression of globin is realized in protein level, the length of the vector is substantially reduced, the lentivirus packaging efficiency is remarkably improved, the complexity and quality control requirements of a production process are lowered, the problems of low efficiency and high energy consumption of the globin lentivirus vector are solved fundamentally, and an innovative scheme is provided for high-cost-performance large-scale stable production of gene therapeutic drugs.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a globin gene double-expression lentiviral vector and an application thereof, in particular to a HBB and HBG2 double-expression lentiviral vector and an application thereof. Background technique [0002] The currently ongoing international thalassemia gene therapy trials are mainly led by BlueBird. The lentiGlobin, a lentiglobin drug produced by the company based on the BB305 vector, has entered Phase II and Phase III clinical trials, and the results are encouraging. HGB-204 and HGB -205, etc. have a significant therapeutic effect on thalassemia. The main process of the treatment plan is: first collect the patient's own hematopoietic stem cells, after in vitro lentivirus infection, the hematopoietic stem cells integrated with the correct globin expression module are returned to the patient, and the HBB globin gene can be restored after stable hematopoietic reconstruction expression. ...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/867C12N5/10A61K48/00A61K38/17A61P7/06
CPCC07K14/005C07K14/47C12N15/86C12N5/0647A61K48/005A61K48/0008A61P7/06C12N2770/32022C07K2319/00C12N2740/15043C12N2800/107C12N2800/22C12N2510/00A61K38/00
Inventor 张磊王文天李慧媛池颖付荣凤鞠满凯孙婷
Owner INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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