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Site specific repairing carrier system and method of blood coagulation factor genetic mutation

A technology for coagulation factors and mutation sites, which can be used in vectors, gene therapy, genetic engineering, etc., and can solve the problems of huge TALENs system, deletion mutation, repeated mutation, ectopic mutation, unsuitable gene defect, etc.

Inactive Publication Date: 2016-05-11
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method can repair blood coagulation factor F8 to a certain extent by means of in situ repair, however, this method needs to import multiple complete genes (i.e. exons 23-26), which still requires an overly large TALENs system, and does not Not suitable for other genetic defects, such as deletion mutations, duplication mutations, ectopic mutations, etc.
In addition, due to the difference in the main types of mutations in blood coagulation factors F9 and F8, only about 40-50% of F8 gene mutations are inversions of coloring question 22 under natural conditions, while F9 mutations are mainly single or multiple single mutations. Point mutation of base combination, so this method is not suitable for in situ repair of coagulation factor F9

Method used

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  • Site specific repairing carrier system and method of blood coagulation factor genetic mutation
  • Site specific repairing carrier system and method of blood coagulation factor genetic mutation
  • Site specific repairing carrier system and method of blood coagulation factor genetic mutation

Examples

Experimental program
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Effect test

Embodiment 1

[0102] Example 1 In situ repair of some bases of the blood coagulation factor IX locus in human HEK293T cells

[0103] 1. Construction of vector pX458-sghF9 targeting human coagulation factor IX

[0104] Schematic diagram of gene in situ repair at FIX site in human cells by CRISPR / Cas system figure 1 shown. Obtain the DNA sequence of human coagulation factor IX from NCBI. In this example, the query sequence is the CDS region of exon 8 of the human coagulation factor IX gene. Look for 5'N in the sense or antisense strand of this CDS sequence 20 NGG3' or 5' CCNN 20 3' sequence. Among them, the first 20 bases N are the bases for complementary pairing between sgRNA and target sequence. The PAM (proto-spacer-ajacent-motif) sequence recognized by s.pyCas9 for the last three bases NGG (CCN) is not included in the sgRNA sequence. Since the promoter driving sgRNA transcription in the pX458 vector is the U6 promoter, a "G" was added to the 5' end of the sgRNA sequence to ensure t...

Embodiment 2

[0113] Example 2, repairing the missing FIX gene fragment in the hemophilia mouse model in vivo by gene in situ repair.

[0114] 1. Information about hemophilia B mice

[0115] In this example, the hemophilia B mice carry a Y381D homozygous mutation in the FIX gene.

[0116] 2. Selection of sgRNA target

[0117] The selection method is the same as the selection of sgRNA in Example 1. The selection of sgRNA in this embodiment is:

[0118] sgmf9-forcaccggAACTCTAAGGTCCTGAAGAA,

[0119] sgmf9-RevaaacTTCTTCAGGACCTTAGAGTTcc,

[0120] 3. Construction of pX458-sgFIX gene in situ repair vector

[0121] In step 2, sgFIX-For and sgFIX-RevDNA were annealed and respectively ligated into pX458 to construct pX458-sgmf9, as Figure 5 shown.

[0122] 4. Construction of adenovirus gene in situ repair vector

[0123] In Step 2, sgmf9-For and sgmf9-Rev were annealed and connected to the H271pAdeno vector. The homologous recombination repair sequence was designed according to the FIX fact...

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Abstract

The invention discloses a method for carrying out in-situ repairing on blood coagulation factor F8 / F9. The method comprises the following steps: in a target genome sequence, selecting the mutation sites of blood coagulation factor as the gene sites for in-situ repairing; designing the binding sites of nuclease of sgRNA sequence of a CRISPR / Cas system; designing a homologous recombinant repairing donor sequence for in-situ repairing; delivering nuclease protein and / or sgRNA and the nucleotide sequence of the homologous recombinant repairing donor to the gene sites of in-situ repairing by a delivering carrier; generating damages to the genome DNA by the nuclease on the gene in-situ repairing sites; and inserting the homologous recombinant repairing donor sequence into the gene in-situ repairing sites so as to repair the gene or supplement the expression of gene. The in-situ repairing of mutation sites of blood coagulation factor can be applied to the clinic, and the method has the advantages of precise induction, safer and controllable process, and definite target.

Description

technical field [0001] The invention relates to the technical field of blood coagulation factor gene editing, in particular to an in situ repair method of blood coagulation factor and its application. Background technique [0002] The human coagulation process is a cascade reaction involving various coagulation factors (such as coagulation factors I, II, VIII, IX, X, XI), etc., any mutation of any factor in this reaction may lead to a cascade reaction The reaction cannot be carried out, and the coagulation process cannot be completed. Clinically, it is manifested as hemophilia symptoms such as coagulation disorders and spontaneous bleeding (especially in joints, muscles and soft tissues.). The vast majority of hemophilias are caused by mutations in genes expressing coagulation factors VIII (FVIII or F8) or IX (FIX or F9). Among them, the coagulation disorder caused by mutations in the VIII gene is called hemophilia A. The VIII gene encodes coagulation factor VIII, a glycop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861A61K48/00A61P7/04
CPCA61K48/005C12N15/86C12N2710/10041C12N2810/40
Inventor 李大力关玉婷王立人刘明耀
Owner EAST CHINA NORMAL UNIV
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