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Compositions, medicaments and sgRNAs for increasing coagulation activity in hemophilia b patients

A technology of hemophilia and composition, applied in the direction of recombinant DNA technology, DNA/RNA fragments, drug combination, etc., can solve the problem of high price, reduce toxic side effects, reduce the dose of virus vector, improve completeness and stability Effect

Active Publication Date: 2021-01-15
成都金唯科生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Infusion of coagulation factor IX preparations (usually recombinantly expressed IX protein in vitro) prophylaxis or on-demand replacement therapy is currently the standard therapy for hemophilia in high-income countries, but it requires frequent infusions and is expensive

Method used

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  • Compositions, medicaments and sgRNAs for increasing coagulation activity in hemophilia b patients
  • Compositions, medicaments and sgRNAs for increasing coagulation activity in hemophilia b patients
  • Compositions, medicaments and sgRNAs for increasing coagulation activity in hemophilia b patients

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Construction of a vector targeting the intron 1 region of the Albumin gene and capable of directional integration of functional coagulation factor IX (hereinafter referred to as F9)

[0053] 1. Strategies for directional integration of functional coagulation factor IX in the intron 1 region of the Albumin gene using the CRISPR / Cas9 gene editing system figure 1 shown.

[0054] 2. sgRNA gene editing efficiency verification

[0055] 2.1 According to the sequence of the intron 1 region of the Albumin gene, sgRNAs targeting different sites in the intron 1 region of the Albumin gene were designed. The sequences of the target sites targeted by each sgRNA are shown in Table 1.

[0056] Table 1

[0057]

[0058] 2.2 According to the target site sequence in Table 1, synthesize each sgRNA oligonucleotide single strand (see Table 2), add 4 bases to the 5' end of the sense and antisense strands during synthesis, and make it compatible with the pX330 vector (vector schematic dia...

experiment example 1

[0080] Detect the expression level of F9 treated with the above-mentioned AAV8.CRISPR-SpCas9 vector system

[0081] Experimental group:

[0082]Targeted therapy group: inject the first AAV virus vector AAV8.SpCas9 (1 × 10 12 genome copy / mouse) and the second AAV viral vector AAV8.sgRNA.donor (3×10 12 Genome copies / mouse) of 8-week-old female hemophilia B mice (n=5);

[0083] Non-targeting control group: AAV8.SpCas9 (1×10 12 Genome copy / mouse) and AAV8.control.donor (empty vector, no expression of sgRNA, 3×10 12 Genome copies / mouse) of 8-week-old female hemophilia B mice (n=5);

[0084] Untreated group: 8-week-old female hemophilia B mice without any treatment (n=5);

[0085] Blood samples were taken at different time points after treatment to detect the expression level and function of F9.

[0086] Detection method: Take blood from the orbit and dilute it with 3.2% sodium citrate anticoagulant 10:1, then centrifuge to get the supernatant plasma, divide it into two parts,...

experiment example 2

[0089] The efficiency of targeted integration of the F9 gene after treatment in hemophilia B mice was examined.

[0090] The experimental grouping is the same as in Experimental Example 1.

[0091] After the treatment, the liver lobe was washed with pre-cooled PBS, absorbed to remove water, placed in OCT embedding medium, frozen in liquid nitrogen, sliced ​​with a cryostat (section thickness 8 μm), fixed, and processed with goat anti-human F9 antibody. Immunofluorescence staining, using ImageJ software to take pictures and analyze the experimental results. Liver homogenate was collected, tissue RNA was extracted, cDNA was synthesized by reverse transcription, target-specific qPCR primers were designed, and Sybr Green qPCR kit was used for fluorescent quantitative PCR detection, and Albumin-specific qPCR was used as a control.

[0092] After the experiment, the liver tissue was harvested, the whole genomic DNA was extracted by homogenization, the genomic DNA was treated with X...

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Abstract

The invention discloses a composition for improving the coagulation activity of a patient with type B hemophilia, a medicine and sgRNA (small guide Ribonucleic Acid), and relates to the field of genetreatment of hemophilia. The composition comprises a first AAV (Adeno-associated Virus) vector and a second AAV vector for expressing the sgRNA; a core sequence of the sgRNA is selected from SEQ ID NO. 6 to SEQ ID NO. 9. The composition can be used for treating the type B hemophilia and recovering the coagulation activity.

Description

technical field [0001] The present invention relates to the field of hemophilia gene therapy, in particular to a composition, medicine and sgRNA for improving blood coagulation activity of hemophilia B patients. Background technique [0002] Hemophilia B is a monogenic X-chromosome-linked recessive hereditary disease of the coagulation system. Hemophiliacs often develop from childhood. Because children are active and active, the probability of joint bleeding is high. Long-term attacks can affect the growth and development of bones and joints. Cause joint deformity and muscle atrophy. Blood coagulation factor IX is deficient in patients with hemophilia B. The protein encoded by blood coagulation factor IX is one of the serine proteases involved in the blood coagulation system. Studies have found that restoring the function of blood coagulation factor IX to ≥1% of its normal activity will greatly improve the clinical manifestations of severe patients. type. Infusion of coagu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/864C12N15/90C12N15/113A61K48/00A61K38/48A61P7/04
CPCA61K38/4846A61K48/005A61P7/04C12N9/644C12N15/113C12N15/86C12N15/907C12N2310/10C12N2750/14143C12N2800/107C12N2810/10C12Y304/21022C12N2310/20
Inventor 杨阳魏于全
Owner 成都金唯科生物科技有限公司
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