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Tissue specific promoter and application thereof

A tissue-specific, promoter technology, applied in applications, tissue culture, specific peptides, etc., can solve the problems of low transfection efficiency of F8 virus vector, production of antibodies and inhibitor reactions, low protein secretion and function, etc., to reduce immunity. Risk of rejection, efficacy of protection, effect of low antibody response

Pending Publication Date: 2022-02-11
BEIJING MEIKANG JIMIAN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current methods of gene therapy using the F8 gene with B domain deletion (F8-BDD) have problems such as low protein secretion and function, low transfection efficiency of F8 viral vectors, and antibody and inhibitor reactions (immune rejection).

Method used

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  • Tissue specific promoter and application thereof
  • Tissue specific promoter and application thereof
  • Tissue specific promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] In this embodiment, a lentiviral vector is constructed, which carries the specific promoter of the present invention and the F8 gene, and specifically includes the following steps:

[0063] (1) Schematic diagram of the structure of the lentiviral vector pEGWI as shown in figure 1 As shown, the wild-type 5' splice donor site was mutated, the enhancer in U3 was deleted, and the silencer (CH4 silencer) was added in U3. For the specific transformation method, please refer to "Contributions of Viral Splice Sites and cis-Regulatory Elements toLentivirus Vector Function, Cui et al. Journal of Virology, July 1999, p.6171–6176”;

[0064] (2) Insertion of different tissue-specific promoters and the F8-BDD gene:

[0065] Whole-gene synthesis of Wasabi gene sequence (expressing fluorescent protein), F8 gene (F8-BDD) sequence (SEQ ID NO:5) with B domain deletion, and tissue-specific promoters EF1α (SEQ ID NO:6), VEC (SEQ ID NO:1), KDR (SEQ ID NO:2), ITGA (SEQ ID NO:3) and the nucl...

Embodiment 2

[0069] In this example, the lentiviral vector constructed in Example 1 was further packaged, purified, and concentrated to obtain a recombinant lentivirus. For the experimental method, refer to ([1] Chang L J, Urlacher V, Iwakuma T, et al. Efficacy and safety analyzes of a recombinant human immunodeficiency virus type 1derived vectorsystem[J].Gene Therapy,1999,6(5):715-728.[2]Chang L J,Zaiss A K.Chang,LJ and Zaiss,AK.Lentiviral vectors.Preparation and use.Methods Mol Med 69:303-318[J].Methods in molecular medicine,2002,69:303-318.)

[0070] For specific steps, please refer to the above documents, and a brief description is as follows:

[0071] (1) The lentiviral vector constructed in Example 1 and the packaging helper plasmid pNHP and pHEF-VSV-G were co-transfected into mammalian cells HEK293T and cultured for 48 hours, and the supernatant viral vector was collected;

[0072] (2) Purify and concentrate the lentivirus obtained from the cultivation to obtain the recombinant len...

Embodiment 3

[0075] In this example, the recombinant lentivirus containing different promoters and Wasabi gene prepared in Example 2 was used for in vitro testing, and the specificity of the promoter in different cells was detected by detecting the amount of fluorescent protein expressed by the Wasabi gene.

[0076] The five lentiviruses (LV-EF1α-Wasabi, LV-VEC-Wasabi, LV-KDR-Wasabi, LV-ITGA-Wasabi and LV-Gp-Wasabi) prepared in Example 2 carrying the normal Wasabi gene were respectively transfected into the endothelium Cell (EC), megakaryocyte (Megakaryocyte) two cell lines, lentiviral transfection method is:

[0077] DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution was added to a six-well plate (Corning, USA), and 3 × 10 4 endothelial cells, or 1×10 5 megakaryocytes at 37°C, 5% CO 2 Cultured under conditions for 18 h, transfected with lentivirus at MOI=200, and supplemented polybrene (Polybrene, 8 μg / mL, Sigma-Aldrich) to a final medium volume of 600...

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Abstract

The invention relates to a tissue specific promoter and application thereof, wherein the nucleic acid sequence of the tissue specific promoter comprises more than 80% of sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. The tissue specific promoter provided by the invention can start specific expression of coding genes in endothelial cells or megakaryocyte-platelet cells, can be applied to gene therapy requiring specific expression genes in endothelial cells or megakaryocyte-platelet cells, ensures the therapeutic effect, reduces the risk of immunological rejection, and saves the therapeutic cost.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a tissue-specific promoter and its application. Background technique [0002] Gene therapy (gene therapy) refers to the introduction of exogenous normal genes into target cells to correct or compensate for diseases caused by defects and abnormal genes, so as to achieve the purpose of treatment, but non-specific expression often occurs in clinical practice, that is, exogenous genes in the human body It is widely expressed and can cause immune rejection in the human body, which is one of the more difficult problems to overcome clinically. [0003] For example, hemophilia A (Hemophilia A, HA), also known as hereditary antihemophilic globulin deficiency or FⅧ (F8) deficiency, is a genetic disorder caused by the gene of coagulation factor VIII (FⅧ gene or F8 gene). For coagulation disorders caused by defects, the current treatment for HA mainly includes protein replacement therapy and gene...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/12C12N15/867C12N5/10C12N7/01A61K48/00A61P7/04
CPCC12N15/113C12N15/86C12N7/00C12N5/0647C07K14/755A61K48/0058A61P7/04C12N2740/15021C12N2740/15043C12N2510/00C07K14/71C07K14/70546C07K14/47C12N2740/16043C12N2830/008A61K48/005
Inventor 张隆基宫洁
Owner BEIJING MEIKANG JIMIAN BIOTECH CO LTD
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