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Virus vector for expressing recombinant human beta-globin and application of virus vector

A technology of recombinant virus and globin, applied in the field of biomedicine, can solve the problem of cost-limiting the clinical application of gene therapy technology

Active Publication Date: 2020-06-05
CARBIOGENE THERAPEUTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

High costs will inevitably limit the future clinical application of gene therapy technology
In addition, too high copy number in a single cell can easily increase the risk of insertion mutations in the genome, causing safety issues [Hacein-Bey-Abina, S., et al., LMO2-associated clonalT cell proliferation in two patients after gene therapy for SCID-X1. Science, 2003.302(5644): p.415-9.]

Method used

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  • Virus vector for expressing recombinant human beta-globin and application of virus vector
  • Virus vector for expressing recombinant human beta-globin and application of virus vector
  • Virus vector for expressing recombinant human beta-globin and application of virus vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Embodiment 1, the determination of HBB expression module gene sequence

[0109] 1. Optimization of HBB expression cassette sequence

[0110] The mRNA sequence (accession number: NM_000518.5) and gene sequence (GeneID: 3043) of natural human HBB were obtained from the NCBI website database (https: / / www.ncbi.nlm.nih.gov / ). The natural HBB expression frame (exon) sequence was extracted from the NM_000518.5 sequence, and the sequence was codon-optimized on the website http: / / sg.idtdna.com / site to obtain the gene-optimized HBB exon sequence. The optimized HBB exon sequence of the gene was used to replace the natural human HBB exon sequence, and at the same time, the 374 bp sequence between the PstI sites in the natural human HBB intron II was deleted to obtain an optimized HBB expression cassette. The polynucleotide sequence of the optimized HBB expression cassette is as 2046-3277 of SEQ ID No.1, 2944-4175 of SEQ ID No.2, 1486-2717 of SEQ ID No.3 or SEQ ID The 308th-1539th...

Embodiment 2

[0129] Example 2, construction and virus packaging of lentivirus and adeno-associated virus vectors overexpressing HBB

[0130] Add EcoRV and XbaI restriction sites at the ends of the above HBB0, HBB1, HBB2, and HBB3 sequences, respectively, and connect them to the lentiviral vector pLV-eGFP (Addgene) ( Figure 4 ) between the EcoRV and XbaI sites to obtain pLV-HBB0, pLV-HBB1, pLV-HBB2, and pLV-HBB3. In addition, a NotI restriction site was added at the end of the HBB0, HBB1 and HBB3 sequences, and connected to the adeno-associated virus vector pAAV-MCS ( Figure 5 ), pAAV-HBB0, pAAV-HBB1, and pAAV-HBB3 were obtained. The above ligation product was transformed into competent Escherichia coli (DH5α). Use Qiagen's endotoxin-free plasmid purification kit to extract and purify the plasmid, and the obtained plasmid can be used for lentivirus and adeno-associated virus packaging experiments after being verified by sequencing. In addition, pLV-eGFP and pAAV-GFP, which replaced the...

Embodiment 3

[0141] Embodiment 3, virus titer determination

[0142] For lentivirus, the virus titer was detected by flow cytometry, while for adeno-associated virus, the virus titer was detected by Q-PCR. The difference is that the viral titer of lentivirus is usually expressed by the number of active units per milliliter of virus fluid (TU / ml), while adeno-associated virus is usually expressed by the number of virus physical particles per milliliter of virus fluid (v.g / ml).

[0143] The specific experimental operation method of virus titer determination of lentivirus :

[0144] 1. HT1080 cells according to 1.5×10 5 Cells / 3ml / well were seeded into 6-well plates. Incubate at 37°C for 24h.

[0145] 2. Aspirate and discard the original medium, add 0.5ml DMEM complete medium (containing 8μg / ml polybrene) containing 5% FBS, and add concentrated pLV-eGFP virus solution dropwise according to the gradient of 0, 6.25, 12.5, 25, 50, and 100μl . Incubate at 37°C for 1 hour, add 2ml of culture...

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Abstract

The invention discloses a virus vector for expressing recombinant human beta-globin and application of the virus vector. The invention provides a DNA fragment A which is formed through linking of thefollowing components from a 5' terminal to a 3' terminal: three DNA enzyme I hypersensitive sites, an HBB promoter, a translation enhancer, an HBB gene expression frame and an HBB transcription enhancer. An HBB expression vector provided by the invention has the capability of efficiently expressing exogenous HBB proteins and has extremely high application values in treatment of beta-thalassemia and sickle cell anemia.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a virus vector for expressing recombinant human beta-globin and its application. Background technique [0002] Hemoglobinopathy (hemoglobinopathy) is a group of hereditary blood diseases caused by abnormal hemoglobin molecular structure (abnormal hemoglobin disease) or abnormal globin peptide chain synthesis (globin anemia), including β-thalassemia, sickle cell anemia etc. 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). , F.B., The Present and Future Global Burden of the Inherited Disorders of Hemoglobin. Hematol Oncol Clin North Am, 2016.30(2):p.327-41.]. β-thalassemia has a high incidence in the southeast and southwest of my country, but is relatively rare in the north, so it is also called thalassemia. The molecular pathology of β-thalassemia is quite...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/867C12N15/864C12N7/01C07K14/805A61K38/42A61P7/06
CPCA61K38/00A61P7/06C07K14/805C12N7/00C12N15/86C12N2740/15021C12N2740/15043C12N2750/14121C12N2750/14143C12N2800/22
Inventor 朱建高杨文君黄翔
Owner CARBIOGENE THERAPEUTICS CO LTD
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