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Product for treating hemophilia A and application thereof

A hemophilia, recombinant virus technology, applied in applications, blood diseases, gene therapy, etc., can solve problems such as the inability to effectively control the expression of BDDFVIII

Active Publication Date: 2021-03-30
济南赛尔生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The promoter sequence used in this patent is EF1α, which can increase the expression of BDD FVIII gene in cells, but EF1α is not a tissue-specific promoter and cannot effectively control the expression of BDD FVIII in vivo

Method used

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  • Product for treating hemophilia A and application thereof
  • Product for treating hemophilia A and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1, the construction of lentiviral vector

[0068] 1. Use restriction enzymes XcmI and SalI to excise the nucleotide sequence of the PGK promoter and GFP gene on the pRRLSIN.cPPT.PGK-GFP.WPRE vector plasmid (Plasmid#12252), and recover the vector backbone;

[0069] 2. Chemical synthesis and / or overlap extension PCR to obtain nucleotide sequence composition 1 (SEQ ID NO.1) comprising PF4 promoter (SEQ ID NO.1) and BDD FVIII (SEQ ID NO.4 or SEQ ID NO.17) .32) or nucleotide sequence composition 2 (SEQ ID NO.33), the obtained nucleotide sequence composition is respectively connected with the vector backbone obtained in step 1 to obtain two kinds of recombinant vectors: pRRL-PF4-BDD FVIII -WPRE-1 and pRRL-PF4-BDD FVIII-WPRE-2, the basic structures of the two vectors are as follows figure 1 As shown, the two only differ in sequence at the position of BDD FVIII.

Embodiment 2

[0070] Embodiment 2, packaging and purification of lentiviral vector

[0071] 1. Taking a 10cm culture dish as an example, plant HEK293T cells in DMEM medium containing 10% fetal bovine serum; wait for the cells to grow to a confluence of 70-80%, and start packaging the virus;

[0072] 2. Discard the original medium, add 50% volume of fresh medium, and continue to incubate;

[0073] 3. The mass ratio of pRRL-PF4-BDD FVIII-WPRE-1 / 2:psPAX2:PMD2.0G is 8:6:2, and the lentiviral vector is packaged by calcium transfer method;

[0074] 4. After 12 hours, change the liquid;

[0075] 5. After 36 hours, collect the cell suspension, filter through a 0.44 μm filter membrane, and centrifuge at 50,000 g for 2 hours;

[0076] 6. Collect the precipitate, which is the lentiviral vector loaded with PF4-BDD FVIII, wherein, the lentiviral vector-1 comprising SEQ ID NO.32, and the lentiviral vector-2 comprising SEQ ID NO.33.

Embodiment 3

[0077] Embodiment 3, preparation of genetically modified Dami cells

[0078] 1. Culture Dami cells (megakaryocytes) in 1640+10% FBS+L-Glutamin medium to logarithmic growth phase;

[0079] 2. Aspirate cells, wash, use X-VIVO10+10%FBS+4ug / ml Polybrene medium to blow and mix Dami cells, press 2×10 6 / well planted at 20 μg / cm 2 In the six-well plate coated with RetroNectin, the cells were divided into 3 groups, group 1 was the control group without adding lentiviral vector; group 2 was transfected with lentiviral vector-1; group 3 was transfected with lentiviral vector-2 Group.

[0080] 3. After 2 hours, according to MOI=5-50, add the lentiviral vector loaded with PF4-BDD FVIII to the cell suspension; add the same amount of lentiviral vector again after 24 hours;

[0081] 4. After 24 hours, absorb the cells, wash, centrifuge, add 1640+10% FBS+L-Glutamin culture medium, adjust it to 1×10 6 / hole was planted in a new six-hole plate; continued to cultivate for 48h;

[0082] 5....

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Abstract

The invention discloses a product for treating hemophilia A and an application thereof. The product comprises an expression cassette of a BDD FVIII gene, a recombinant vector containing the expressioncassette, a recombinant virus, especially a recombinant lentivirus, a recombinant bacterium or a recombinant cell. A promoter of the BDD FVIII gene in the expression cassette comprises a PF4 promoter. According to the lentiviral vector containing the PF4 promoter and the BDD FVIII gene expression cassette, the BDD FVIII gene can be specifically expressed in platelets formed by cytoplasm sheddingof megakaryocytes derived from CD34+ cells through the PF4 promoter, so that the blood coagulation function is achieved, and the lentiviral vector can be applied to gene therapy of hemophilia A of allages.

Description

technical field [0001] The invention relates to the field of gene therapy, in particular to a product and application for treating hemophilia A. Background technique [0002] Hemophilia is an X-chromosome-linked recessive hereditary bleeding disorder, divided into two types: hemophilia A (hemophilia A) and hemophilia B (hemophilia B). Hemophilia A is a deficiency of blood clotting factor VIII (FVIII) caused by a genetic mutation. The FVIII gene is located at the end of the long arm of the X chromosome: (Xq28), which encodes a peptide chain and is arranged in the form of A1-A2-B-A3-C1-C2. The main mutation types closely related to the development of inhibitors in patients with severe hemophilia include large deletions, nonsense mutations, and intron 22 inversions, followed by small deletions and insertions, and missense mutations. There are no population or regional differences in the incidence of hemophilia. In the male population, the incidence of hemophilia A is about 1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10A61K48/00A61K38/37A61P7/04
CPCC12N15/86A61K48/005A61K38/37A61P7/04C12N2740/15043
Inventor 罗昀宋珂慧郭栋邢晓郭伟
Owner 济南赛尔生物科技股份有限公司
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