Codon-optimized blood coagulation factor VIII gene and construct comprising codon-optimized blood coagulation factor VIII gene

A codon optimization and coagulation factor technology, applied in the field of coagulation factor VIII gene and its constructs, can solve problems such as affecting gene transcription efficiency

Active Publication Date: 2020-10-23
KANGLIN BIOTECHNOLOGY (HANGZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, among different general codon optimization principles, the existing technology and knowledge believe that excessive GC content will affect the transcription efficiency of genes, so avoiding excessive GC content is a repeatedly mentioned optimization principle

Method used

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  • Codon-optimized blood coagulation factor VIII gene and construct comprising codon-optimized blood coagulation factor VIII gene
  • Codon-optimized blood coagulation factor VIII gene and construct comprising codon-optimized blood coagulation factor VIII gene
  • Codon-optimized blood coagulation factor VIII gene and construct comprising codon-optimized blood coagulation factor VIII gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Embodiment 1 coagulation factor VIII gene expression framework design

[0074] Based on the amino acid sequence of recombinant human coagulation factor VIII for injection (Ren Jie Xyntha) produced by Pfizer Pharmaceuticals, the expression framework was designed as the wild-type expression framework. The sequence mainly includes A1, A2, B, a3, A3, C1, and C2 regions , in which 887 amino acids are deleted after the B domain is truncated, and only 21 amino acids are contained, and the expression frame number is P0001.

[0075] On the basis of the expression framework P0001, three other expression frameworks P0002, P0003, and P0004 were designed to optimize codons for different conditions. The optimization principle is to preferentially select mammalian cell-biased codons, while avoiding hidden splicing sites, splicing donor and acceptor sequences, immature tailing signals, strong mRNA secondary structures, RNA unstable sequences, Transcription termination signal, etc.

...

Embodiment 2

[0079] Example 2 Construction of coagulation factor VIII gene expression lentiviral vector

[0080] The coagulation factor VIII gene expression framework P0001 designed in Example 1 was driven by the TTR promoter, and the other expression frameworks were respectively driven by the TTR and EF1a promoters, and then cloned into the lentiviral backbone to form a lentiviral vector. The lentiviral backbone was derived from Kanglin Biotechnology (Hangzhou) Co., Ltd.'s own HIV-1-based third-generation replication-deficient self-inactivating pseudo-enveloped lentiviral backbone—pKL-CCL, nucleotide sequence SEQ ID NO:01, Atlas to show opinions image 3 . The lentiviral backbone contains chimeric LTR promoter, HIV-1 packaging signal (ψ), central polypurine region (cPPT), Rev response element (RRE), polypurine fragment (PPT), woodchuck hepatitis B virus post-transcriptional Regulatory element (WPRE), SV40 virus polyadenylation signal (SV40pA signal), SV40 virus replication initiation si...

Embodiment 3

[0105] Example 3: Coagulation factor VIII gene expression lentiviral packaging

[0106] The coagulation factor VIII gene lentiviral expression vector (pKL-CCL-TTR-P0001, pKL-CCL-TTR-P0002, pKL-CCL-TTR-P0003, pKL-CCL-TTR-P0004, pKL-CCL -EF1a-P0002, pKL-CCL-EF1a-P0003, pKL-CCL-EF1a-P0004), envelope plasmid (pKL-Kan-Vsvg, its nucleotide sequence is shown in SEQ ID NO: 17) and packaging plasmid ( pKL-Kan-Rev, whose nucleotide sequence is shown in SEQ ID NO: 18, pKL-Kan-GagPol, whose nucleotide sequence is shown in SEQ ID NO: 19) co-transfected 293T cells (purchased from the U.S. Type strain collection center (ATCC), preservation number is CRL-3216), in the 293T cell line, carry out the packaging of the coagulation factor VIII gene therapy lentiviral vector. The transfection method is the transient transfection of eukaryotic cells mediated by PEI cationic polymer, the PEI cationic polymer is the PEI-Max transfection reagent purchased from Polysciences (purchased from Polysciences,...

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Abstract

The invention relates to the field of biological medicine, in particular to a codon-optimized blood coagulation factor VIII gene and a construct comprising the codon-optimized blood coagulation factorVIII gene. The codon-optimized blood coagulation factor VIII gene is provided, a GC content in a sequence of the blood coagulation factor VIII gene is at least 54%, a CpG island number is at least 122, and a gene sequence of a B domain in the codon-optimized blood coagulation factor VIII gene is a truncated sequence. The nucleic acid construct is further provided, and the nucleic acid construct comprises the codon-optimized blood coagulation factor VIII gene. The codon-optimized blood coagulation factor VIII gene and the construct comprising the codon-optimized blood coagulation factor VIII gene have the following beneficial effects that the expression quantity of a blood coagulation factor VIII is significantly improved; the activity level of the blood coagulation factor VIII is significantly raised; and a dosage is low, immunoreaction and the immunogenicity are reduced, and risks are relatively controllable.

Description

technical field [0001] The invention relates to the technical field of medicine, in particular to a codon-optimized blood coagulation factor VIII gene and a construct thereof. Background technique [0002] Platelet coagulation factors are various protein components involved in the blood clotting process. The common function of these factors is to maintain normal coagulation physiological function in bleeding state. Deficiency of one or more clotting factors results in a group of inherited bleeding disorders called hemophilia. Among them, the most common clinical hemophilia A is caused by coagulation factor VIII-antihemophilic globulin A (AHGA), which is an X-linked recessive genetic disease. Patients presented with coagulopathy of varying severity and spontaneous bleeding. [0003] Coagulation factor replacement therapy is currently the only confirmed and effective drug for the treatment of hemophilia A, which is not only expensive, but also produces inhibitory factors af...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/755C12N15/867C12N7/01C12N5/10A61K38/37A61P7/04C12R1/93C12R1/91
CPCA61K38/37A61P7/04C07K14/755C12N7/00C12N15/86C12N2740/15021C12N2740/15043C12N2800/107C12N2800/22
Inventor 吴昊泉党颖苏玲玲叶青牛琦
Owner KANGLIN BIOTECHNOLOGY (HANGZHOU) CO LTD
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