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Application of lentiviral vector in preparation of medicine for treating beta-thalassemia

A lentiviral vector and thalassemia technology, applied in the field of genetic engineering, can solve the problems of low titer of toxin production, bulky structure, high production cost, etc., and achieve the effect of increasing virus production, improving expression efficiency, and reducing the risk of cancer

Active Publication Date: 2022-05-10
GENMEDICN BIOPHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Technical problem to be solved: In view of the above technical problem, the present invention provides the application of lentiglobin vector in the preparation of drugs for the treatment of β-thalassemia, which can effectively solve the bloated structure of the above-mentioned LentiGlobin virus vector, low titer of toxin production, high production cost, β-thalassemia - Deficiencies such as suboptimal globin expression levels

Method used

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  • Application of lentiviral vector in preparation of medicine for treating beta-thalassemia
  • Application of lentiviral vector in preparation of medicine for treating beta-thalassemia
  • Application of lentiviral vector in preparation of medicine for treating beta-thalassemia

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Experimental program
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Effect test

Embodiment 1

[0030] 1. Lentiviral vector pCCL-SIN-cPPT-LCR2.7K-pHBB-CI-β A-T87Q- Construction of RbPA, including vector pCCL-SIN-cPPT-MCS-RbPA (SEQ ID No: 1), 2.7kb β-LCR (LCR2.7K) regulatory sequence (SEQ ID No: 2), β-globin promoter ( pHBB) sequence (SEQ ID No:6), chimeric intron (CI) enhancer sequence (SEQ IDNo:7) and encoding codon-optimized β-globin gene β A-T87Q Sequence (SEQ ID No: 9), and the code at codon 87 contains the mutation T87Q, that is, the mutation from threonine to glutamine.

[0031] 1.1 The company's vector plasmid pCCL-SIN-cPPT-MCS-RbPA (SEQ ID No: 1) was used as the vector for the preparation of lentiviral vector, and it was double digested with restriction endonucleases XhoI and MluI at 36.5-37.5°C 0.8-1.2h, after agarose electrophoresis, the pCCL-SIN-cPPT-MCS-RbPA skeleton fragment was recovered by cutting the gel;

[0032] The LCR2.7K regulatory sequence (SEQ ID No: 2) was amplified by PCR, and the 5' end was added with a protection base and an XhoI restriction ...

Embodiment 2

[0101] 1. Lentiviral vector pCCL-SIN-cPPT-LCR3.6K-pHBB-CI-β A-T87Q - Construction of RbPA, including vector pCCL-SIN-cPPT-MCS-RbPA (SEQ ID No: 1), 3.6kb β-LCR (LCR3.6K) regulatory sequence (SEQ ID No: 3), β-globin promoter ( pHBB) sequence (SEQ ID No:6), chimeric intron (CI) enhancer sequence (SEQ IDNo:7) and encoding codon-optimized β-globin gene β A-T87Q Sequence (SEQ ID No: 9), and the code at codon 87 contains the mutation T87Q, that is, the mutation from threonine to glutamine.

[0102] 1.1 The company's vector plasmid pCCL-SIN-cPPT-MCS-RbPA (SEQ ID No: 1) was used as the vector for the preparation of lentiviral vector, and it was double digested with restriction endonucleases XhoI and MluI at 36.5-37.5°C 0.8-1.2h, after agarose electrophoresis, the pCCL-SIN-cPPT-MCS-RbPA skeleton fragment was recovered by cutting the gel;

[0103] The LCR3.6K regulatory sequence (SEQ ID No: 3) was amplified by PCR, and the 5' end was added with a protection base and an XhoI restriction...

Embodiment 3

[0131] 1. Lentiviral vector pCCL-SIN-cPPT-LCR4.6K-pHBB-CI-β A-T87Q - Construction of RbPA, including vector pCCL-SIN-cPPT-MCS-RbPA (SEQ ID No: 1), 4.6kb β-LCR (LCR4.6K) regulatory sequence (SEQ ID No: 4), β-globin promoter ( pHBB) sequence (SEQ ID No:6), chimeric intron (CI) enhancer sequence (SEQ IDNo:7) and encoding codon-optimized β-globin gene β A-T87Q Sequence (SEQ ID No: 9), and the code at codon 87 contains the mutation T87Q, that is, the mutation from threonine to glutamine.

[0132] 1.1 The company's vector plasmid pCCL-SIN-cPPT-MCS-RbPA (SEQ ID No: 1) was used as the vector for the preparation of lentiviral vector, and it was double digested with restriction endonucleases XhoI and MluI at 36.5-37.5°C 0.8-1.2h, after agarose electrophoresis, the pCCL-SIN-cPPT-MCS-RbPA skeleton fragment was recovered by cutting the gel;

[0133] The LCR4.6K regulatory sequence (SEQ ID No: 4) was amplified by PCR, and the 5' end was added with a protection base and an XhoI restriction...

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Abstract

The invention discloses application of a lentiviral vector in preparation of a medicine for treating beta-thalassemia. The lentiviral vector is composed of a pCCL-SIN-cPPT-MCS-RbPA skeleton, a regulatory sequence A, a promoter sequence B, an enhancer sequence C and a gene sequence D. The lentivirus can enhance the specific expression of genes and reduce the size of the vector, so that the virus packaging efficiency is improved, the potential carcinogenic risk of the virus vector to tested cells is reduced, and the lentivirus vector infects hematopoietic stem cells (CD34 + cells) of beta-thalassemia patients, so that the beta-thalassemia patients are infected with hematopoietic stem cells (CD34 + cells). The expression level of exogenous beta globin (Hb beta A-T87Q) containing 87th amino acid mutation is quantified by high performance liquid chromatography, the exogenous beta globin (Hb beta A-T87Q) can be distinguished from wild beta globin expressed by an endogenous gene or derived from blood transfusion, and high expression of Hb beta A-T87Q is observed.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the application of a lentiviral vector in the preparation of medicines for treating beta-thalassemia. Background technique [0002] β-thalassemia is one of the most common monogenic autosomal recessive genetic diseases in the world, which is mainly caused by the decrease or obstacle of β-globin chain synthesis due to the defect of β-globin gene or its regulatory sequence, which leads to α / An imbalance in the beta-globin ratio is caused by an inability to produce enough hemoglobin. The clinical manifestations of β-thalassemia are closely related to the degree of α / β-globin chain imbalance. most beta 0 / β 0 genotype patients and partial beta E / β 0 Patients with the genotype produce little beta chains and often present with transfusion-dependent beta-thalassemia, with severe clinical symptoms requiring >100 mL / kg of packed RBC (red blood cells) or ≥...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/67C12N15/12A61K48/00A61K38/42A61P7/06
CPCC12N15/86C07K14/805A61K48/0058A61K38/42A61P7/06C12N2740/15043C12N2800/107C12N2800/22C12N2830/008
Inventor 董文吉张艳君刘子瑾董祖伊赵忠亮程谟斌
Owner GENMEDICN BIOPHARMA INC
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