Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

F8 protein variant and gene therapy vector prepared by using same

A protein variant and gene therapy technology, applied in gene therapy, using vectors to introduce foreign genetic material, vectors, etc., to achieve the effects of increasing production titers, reducing the amount of vectors, and improving efficacy and safety

Active Publication Date: 2021-09-03
天津协和生物科技开发有限公司
View PDF6 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expression level of this variant can be increased by 17 times, but the protein level is only increased by 30%

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • F8 protein variant and gene therapy vector prepared by using same
  • F8 protein variant and gene therapy vector prepared by using same
  • F8 protein variant and gene therapy vector prepared by using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1 vector construction

[0072] First, use the KAPAHiFi PCR amplification kit to amplify the BDDF8 sequence; wherein, the BDDF8-N6 nucleic acid sequence (SEQ ID NO.17) is shown; the sequence of the amino acid that is expected to be deleted is directly synthesized into gBlocks at IDT; the plasmid backbone is digested by enzymes and PCR, and then use the NEBuilder HiFi DNA Assembly kit to assemble each fragment.

[0073] All vector sequences were verified using endonuclease and Sanger sequencing to obtain plasmids expressing variants 1 to 13.

Embodiment 2

[0074] Example 2 AAV virus packaging, production, concentration, purification and titer determination

[0075] Utilize AAV three-plasmid packaging system for packaging, the three-plasmid packaging system includes: 1) destination vector pAAV-BDDF8-N6-variant; 2) trans plasmid pAAV2 / 8, containing rep and cap genes; 3) adenovirus helper plasmid pHelper (Cell Biolabs), the ratio of the amount of each plasmid is 2:1:1;

[0076] PEI "Max" is used to transfect 293T cells to produce AAV, and the 293T cells are packaged when the confluence of 293T cells is about 80-90%. The base was concentrated about 50 times, and then the AAV vector was purified by iodixanol density gradient centrifugation.

[0077] The AAV titer was determined by qPCR. The iodixanol-purified virus was diluted 3 times with 0.1×TE+20ng / μL SalmonDNA diluent, and heated at 98°C for 5 minutes. Use diluent 0.1×TE+2ng / μL Salmon DNA to dilute the heated AAV virus, and make 5 consecutive 3-fold (5μL+10μL) dilutions.

[00...

Embodiment 3

[0085] Example 3 F8 Activity Detection

[0086] In this example, the AAV8-BDDF8-N6 variant was used to treat hemophilia A mice and followed up to detect F8 activity. Exon 16 of the F8 gene in hemophilia A mice is knocked out, resulting in the abnormal expression of coagulation factor F8, and the F8 activity test results are usually 6% to 8%, resulting in coagulation disorders.

[0087] AAV8-BDDF8-N6 variant injected into hemophilia A mice as Figure 4 As shown, the hemophilia A mice of 6 to 8 weeks were injected to produce the dominant AAV-F8 variant of the present invention, injected uniformly through the tail vein, and the injection dose was 2E+12vg / kg, and the mice were observed every week after injection. The physical condition of the mice was monitored, and blood was collected from the mice at 4, 6, 10, and 20 weeks after injection to detect the activity of F8.

[0088] (1) Blood collection and plasma separation from hemophilia A mice

[0089] After AAV tail vein injec...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides an F8 protein variant and a gene therapy vector prepared by using the same. The F8 protein variant is any one of amino acid sequences obtained by deleting at least four amino acids from a BDDF8-N6 variant, wherein the deleted amino acids contain furin recognition sites RHQR. According to the F8 protein variant provided by the invention, a B structural domain is optimized on the basis of the BDDF8-N6 variant, the furin recognition sites and nearby amino acids of the F8 protein are deleted, a novel efficient variant is obtained, the titer of the obtained variant vector is increased, the activity of F8 in an in-vivo test is relatively high, and the F8 protein variant has a relatively high clinical transformation value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a F8 protein variant and a gene therapy carrier prepared by using the same, in particular to a F8 protein variant obtained by optimizing the B domain and the development and construction of a gene therapy carrier use. Background technique [0002] Hemophilia A (HA) is a monogenic hereditary hemorrhagic disease caused by the loss of function of the blood coagulation factor F8 gene, which is life-threatening in severe cases. Infusion of F8 protein is currently the main treatment, requiring 2-3 weekly intravenous injections, poor quality of life, and expensive treatment. Gene therapy is the only cure for hemophilia. [0003] The preferred vector for hemophilia A gene therapy is AAV vector. After more than 20 years of research, hemophilia A gene therapy delivered by AAV vector has entered clinical trials. Among them, the valoctocogene roxaparvovec (BMN270, NCT02576795) ge...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K14/755C12N15/12C12N15/864A61K48/00A61K38/37A61P7/04G01N33/68
CPCC07K14/755C12N15/86A61K48/0058A61K48/0008A61P7/04G01N33/86G01N33/6854C12N2750/14143C12N2800/107C12N2830/008A61K38/00G01N2333/755
Inventor 程涛张健萍赵梅殷梦迪李斯昂杨智学张凤李国华张孝兵
Owner 天津协和生物科技开发有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com