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A β-globin recombinant lentiviral vector and its application

A technology of recombinant lentivirus and globin, which is applied in the field of molecular biology, can solve the problems of high production cost of vectors and low titer of lentivirus packaging, and achieve the effects of reducing potential carcinogenic risk, low production cost and high packaging titer

Active Publication Date: 2022-07-22
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 problems, the present invention provides a β-globin recombinant lentiviral vector and its application, which can effectively solve the above-mentioned problems of low lentiviral packaging titer and high production cost of the vector

Method used

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  • A β-globin recombinant lentiviral vector and its application
  • A β-globin recombinant lentiviral vector and its application
  • A β-globin recombinant lentiviral vector and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1: Vector Construction

[0039]1. Construction of lentiviral vector pCCL-SIN-cPPT-LCR2.7K-β-globin-Genomic-T87Q-RbPA, including vector pCCL-SIN-cPPT-MCS-RbPA (SEQ ID No: 1), 2.7kb β- LCR regulatory sequence (SEQ ID No:2), β-globin promoter sequence (SEQ ID No:3), genome sequence encoding β-globin (SEQ ID No:4), β-globin polyA sequence (SEQ ID No: 5) and β-globin enhancer sequence (SEQ ID No: 6).

[0040] 1.1 Double-enzyme digestion of the pBSK vector plasmid with restriction enzymes ClaI and XhoI at 36.5-37.5°C for 0.8-1.2h, and after agarose electrophoresis, cut the gel to recover the pBSK vector fragment;

[0041] will express Hbβ A-T87Q The genomic sequence of the expression cassette is divided into β-globin1 and β-globin2 and carry out PCR respectively, wherein the primer sequence PCR1 used for the β-globin1 gene fragment is SEQ ID No:7, PCR2 is SEQ ID No:8, and the β-globin2 gene The primer sequences PCR3 used in the fragment are SEQ ID No: 9, and PCR4 i...

Embodiment 2

[0075] Example 2: Production and purification of lentivirus

[0076]2.1 The successfully constructed pCCL-SIN-cPPT-LCR2.7K-β-globin-Genomic-T87Q-RbPA plasmid and the lentiviral packaging kit plasmid Mix were co-transfected in a ratio of 1:3 and inoculated in a 10-layer cell factory one day before The HEK293T cells were replaced with fresh DMEM medium 6 hours after transfection, and the supernatant was collected after 72 hours for chromatographic purification;

[0077] 2.2 Purification Using a tangential flow filtration-chromatography system, using core700 chromatography and Q ImpRes chromatography purification process, the lentivirus was purified. The specific purification process is as follows:

[0078] ① Benzonase treatment (nuclease digestion): Treat with 25U / mL of Benzonase at 37°C for 1 hour to remove contaminants such as plasmid DNA remaining during transfection and genome released from lysed cells;

[0079] ② MF (clarification): The virus harvest solution is filtered (...

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Abstract

The invention discloses a β-globin recombinant lentiviral vector and an application thereof. The β-globin recombinant lentiviral vector is pCCL-SIN-cPPT-LCR2.7K-β-globin-Genomic-T87Q-RbPA, which is Includes pCCL‑SIN‑cPPT‑MCS‑RbPA backbone, 2.7kb β‑LCR regulatory sequence and β globin Hbβ expressing amino acid mutation at position 87 A‑T87Q The expression cassette of β-LCR is short for β-globin locus control region. The lentiviral vector of the invention has high packaging titer, low vector production cost, reduces the potential carcinogenic risk of the virus vector to the test cells, and more efficiently and stably expresses Hbβ A‑T87Q , providing an important basis for the cure of patients with transfusion-dependent β-thalassemia.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a beta-globin recombinant lentiviral vector and its application. Background technique [0002] Beta-thalassemia is one of the most common single-gene autosomal recessive diseases in the world, and it is a fatal and disabling blood disease that seriously threatens human health. High incidence in West Asia (2-5%), Southeast Asia (up to 10%) and South Asia (up to 18%). In my country, the high-incidence areas are mainly distributed in the provinces and regions south of the Yangtze River, among which the Guangdong and Guangxi regions are the most serious, and with the increase of population mobility, it has shown a trend of spreading to the north. Currently, there are an estimated 200,000 beta-thalassemia patients being treated globally. [0003] At present, the widely implemented clinical treatments for β-thalassemia include standardized blood transfusion, dru...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N15/12C12N7/01A61K48/00A61K38/42A61P7/06
CPCC12N15/86C12N7/00C07K14/805A61K48/005A61K38/42A61P7/06C12N2740/15021C12N2740/15043
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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