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Recombinant blood coagulation factor VIII and application thereof

A technology for coagulation factors and recombinant cells, applied in the directions of coagulation/fibrinolysis factor, factor VII, application, etc., can solve the problems of low protein secretion and function, low transfection efficiency of F8 virus vector, production of antibodies and inhibitor reactions, etc. Achieve low antibody response, improve transfection efficiency and expression efficiency, and correct bleeding phenotype

Active Publication Date: 2021-08-13
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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  • Recombinant blood coagulation factor VIII and application thereof
  • Recombinant blood coagulation factor VIII and application thereof
  • Recombinant blood coagulation factor VIII and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] This embodiment provides a method for constructing a lentiviral vector, which specifically includes the following steps:

[0074] (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 GT is mutated to CA, the enhancer in U3 is deleted, and a silencer (CH4 silencer) is added in U3. For the specific transformation method, please refer to "Contributions of Viral Splice Sites and cis -Regulatory Elements to Lentivirus Vector Function, Cui et al. Journal of Virology, July 1999, p.6171–6176”;

[0075] (2) Insertion of promoter and F8-BDD, F8-BDD-N8 or F8-BDD-299 gene:

[0076] Chemically synthesize normal F8-BDD (sequence shown in SEQ ID NO.7), F8-BDD-N8 (sequence shown in SEQ ID NO.5) through the entire gene and select possible glycosylation site sequences ), F8-BDD-299 (sequence shown in SEQ ID NO.6) gene sequence, and human EF1α promoter sequence added; normal F8-BDD gene structure diagram ...

Embodiment 2

[0080]In this example, the lentiviral vector constructed in Example 1 was further packaged, purified, and concentrated to obtain the 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 vector system[J].Gene Therapy,1999,6(5):715-728.[2]Chang L J,ZaissAK.Chang,LJand Zaiss,AK.Lentiviral vectors.Preparation and use.Methods Mol Med69 :303-318[J].Methods in molecular medicine, 2002,69:303-318.)

[0081] For specific steps, please refer to the above documents, and a brief description is as follows:

[0082] (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;

[0083] (2) purifying and concentrating the lentiviruses collected from the culture to obtain the recombinant...

Embodiment 3

[0086] In this example, the recombinant lentivirus prepared in Example 2 was tested in vitro.

[0087] Three lentiviruses (LV-F8-BDD, LV-F8-BDD-N8 and LV-F8-BDD-N8 and LV-F8-BDD- 299) respectively transfected EA-hy926 endothelial cells, lentiviral transfection methods include:

[0088] Add DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution to a six-well plate (Corning, USA), and inoculate 4 × 10 per well. 4 EA-hy926 cells at 37°C, 5% CO 2 Cultured under conditions for 18 h, transfected with lentivirus at MOI=50, and supplemented polybrene (Polybrene, 8 μg / mL, Sigma-Aldrich) to a final volume of 600 μL of the medium, transfected for 24 h, and then replaced with fresh medium every day, When the confluence of the cells reaches 90%, transfer the cells to T75cm 2 Culture flask (Corning, USA).

[0089] Perform protein expression detection to clarify the expression of F8-BDD, F8-BDD-N8 and F8-BDD-299 genes in cells. The specific process is as fo...

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Abstract

The invention relates to a recombinant blood coagulation factor VIII and application thereof. The B domain of the recombinant blood coagulation factor VIII comprises more than 80% of an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2. The B domain of the recombinant blood coagulation factor VIII is subjected to gene modification, and is rich in glycosylation sites, so that the recombinant blood coagulation factor VIII has an efficient blood coagulation function, is easy to secrete outside cells, is easy to interact with auxiliary factors such as thrombin (FIIa) and the like, is low in antibody reaction, and can effectively guarantee the treatment effect, reduce the immunological rejection risk and save the treatment cost.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a recombinant blood coagulation factor VIII and its application. Background technique [0002] Hemophilia A (Hemophilia A, HA), also known as hereditary antihemophilic globulin deficiency or FⅧ deficiency, is a blood clotting disorder caused by a genetic defect in the coagulation factor VIII gene (FⅧ gene or F8 gene) obstacle. The current treatment for HA is mainly protein replacement therapy (RPT) based on plasma-derived coagulation factors or exogenously cultured recombinant proteins, but PRT has limitations such as short half-life, high cost, and long treatment cycle. [0003] 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 therapeutic purposes. Currently, gene therapy is considered to be the most promising treatment for HA. [0004] Some studies have a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/755C12N15/12C12N15/867C12N7/01C12N5/10A61K38/37A61P7/04
CPCC07K14/755C12N15/86C12N7/00A61P7/04C12N2740/15021C12N2740/15043A61K38/00C12N2740/16043C12N2840/44A61K48/005
Inventor 张隆基宫洁章睿
Owner BEIJING MEIKANG JIMIAN BIOTECH CO LTD
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