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
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expression level of this variant can be increased by

Method used

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  • 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

Example Embodiment

[0071] Example 1 Construction of vector embodiment

[0072] First, KAPAHiFi PCR amplification kit BDDF8 amplified sequence; wherein, BDDF8-N6 nucleic acid sequence (SEQ ID NO.17) as shown; amino acid sequence would be expected to remove the direct synthesis gBlocks IDT Corporation; plasmid backbone by restriction enzyme digestion and obtained by PCR, and then using NEBuilder HiFi DNA assembly kit kit for assembling individual segments.

[0073] All vectors were sequence using the Sanger endonucleases and sequencing verified, to obtain a plasmid expressing the variant ~ XIII variant.

Example Embodiment

[0074] Example 2 AAV viral packaging embodiment, production, concentration, purification, and titer

[0075] AAV plasmids using three packaging system for packaging, said packaging system comprising the three plasmids: 1) destination vector pAAV-BDDF8-N6-variant; 2) trans plasmid pAAV2 / 8, comprising rep and cap genes; 3) the adenovirus helper plasmid pHelper (Cell Biolabs), the ratio between the amounts of each plasmid was 2: 1: 1;

[0076] Use PEI "Max" 293T cells producing AAV, viral packed at about 80 ~ 90% 293T confluent cells; virus was concentrated to a tangential flow filtration system (Tangential flow filtration, TFF), a large volume of culture containing the virus may be yl about 50-fold concentrated, then use iodixanol density gradient centrifugation, purified AAV vector.

[0077] AAV titer was measured using qPCR, iodixanol purified virus using 0.1 × TE + 20ng / μL SalmonDNA diluted 3-fold dilutions, 98 ℃ 5min after the completion of heating. Use dilutions of 0.1 × TE...

Example Embodiment

[0085] Example 3 F8 activity assay

[0086] Examples using AAV8-BDDF8-N6 variants and treatment of hemophilia A mice were followed for detecting activity of the present embodiment F8. No. 16 outer hemophilia A mice F8 gene exon knocked out, resulting in coagulation factors can not express normal F8, F8 activity test results are usually 6% to 8%, and further clotting disorders.

[0087] AAV8-BDDF8-N6 variant hemophilia A mice injected as Figure 4 FIG, 6 to 8 weeks of hemophilia A mice, the advantages of AAV-F8 variants produced according to the present invention an injection, tail vein injection uniform, is injected dose 2E + 12vg / kg, was observed after injection of small week rat physical condition, and 4,6,10,20 weeks after injection mice were bled, respectively, detecting the activity of F8.

[0088] (1) Hemophilia A mice were bled and plasma separation

[0089] AAV after intravenous injection blood was taken at various time points (2 weeks, 4 weeks, 8 weeks, 12 weeks). Hemoph...

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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

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Application Information

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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
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