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Recombinant promoters, vectors and applications thereof for protein expression in the liver

A protein expression and promoter technology, which is applied to animal/human proteins, using vectors to introduce foreign genetic material, vectors, etc., can solve the problems of limited loading capacity, poor expression intensity of liver-specific promoters, etc., and achieve enhanced expression ability Effect

Active Publication Date: 2022-04-08
JINFAN BIOMEDICAL TECH (WUHAN) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In response to the defects or improvement needs of the prior art, the purpose of the present invention is to provide a liver-specific promoter with small size and high expression intensity, and apply it to drive the expression of larger exogenous genes (such as FVIII blood coagulation factor gene), This solves the technical problems of limited loading capacity and poor expression intensity of existing liver-specific promoters in existing rAAV vectors used in gene therapy

Method used

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  • Recombinant promoters, vectors and applications thereof for protein expression in the liver
  • Recombinant promoters, vectors and applications thereof for protein expression in the liver
  • Recombinant promoters, vectors and applications thereof for protein expression in the liver

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] recombinant promoter fragment

[0054] A recombinant promoter fragment consisting of the sequence identified in Table 1 was generated.

[0055] Table 1: Sequences of recombinant promoter fragments

[0056] promoter name third polynucleotide second polynucleotide first polynucleotide AVS1 promoter none none SEQ ID NO.1 AVS2 promoter none SEQ ID NO.2 SEQ ID NO.1 AVS3 promoter none SEQ ID NO.3 SEQ ID NO.1 AVS4 promoter SEQ ID NO.4 SEQ ID NO.2 SEQ ID NO.1 AVS5 promoter SEQ ID NO.5 SEQ ID NO.2 SEQ ID NO.1 AVS6 promoter SEQ ID NO.4 SEQ ID NO.3 SEQ ID NO.1 AVS7 promoter SEQ ID NO.5 SEQ ID NO.3 SEQ ID NO.1

[0057] The first polynucleotide, the second polynucleotide and the third polynucleotide (when present) are contiguously linked in the above-mentioned promoter fragment. The resulting promoters were named AVS1, AVS2, AVS2, AVS3, AVS4, AVS5, AVS6 and AVS7 in sequence.

Embodiment 2

[0059] Comparison of the effect of different promoters on the activation of mCHERRY fluorescent gene in animal experiments

[0060] 1. Construction of rAAV expression vectors including different promoters

[0061] In order to compare the activation strength of different promoters, the present invention constructs the above-mentioned chimeric promoter, the reported broad-spectrum strong promoter CMV and the liver-specific promoter TBG on the AAV vector PFD-rAAV-CMV-mCHERRY-bGHpA, as shown in figure 1 shown.

[0062] figure 1 Middle ITR (inverted terminal repeat), 145bp inverted terminal repeat sequence; CMVenhancer / promoter, human cytomegalovirus early promoter; mCHERRY, red luciferase gene reading frame; BGHpolyA, bovine growth hormone polyadenylation Nucleotide plus microsignal; Amp, reading frame of ampicillin resistance gene; GmR, reading frame of hygromycin resistance gene; Tn7F / R, Tn7 transposon; ori: origin of replication; XhoI, AgeI, SacI, restriction endonuclease si...

Embodiment 3

[0077] Comparison of animal experiments on the effect of different promoters on the activation of FVIII factors

[0078] 1. Construction of BDD-FVIII vectors initiated by different promoters

[0079] In order to further discuss whether the high activation levels of AVS1, AVS2, and AVS3 promoters in the liver are affected by the genes connected downstream, and whether adding expression regulation enhancement elements (AVS4, AVS5, AVS6, AVS7) before AVS2 and AVS3 can further increase the target gene expression. Promoter information is shown in Table 3.

[0080] Table 3 Sequence information of different promoters

[0081]

[0082] The FVIII factor was replaced by the reporter gene mCHERRY, and different promoters, BDD-FVIII (B domain deleted FVIII) and sPolyA expression cassettes were constructed according to the molecular cloning method in Example 2, and constructed into the AAV vector PFD-rAAV-HLP-BDD-FVIII- spolyA( image 3 ), wherein the PFD-rAAV-HLP-BDD-FVIII-spolyA v...

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Abstract

The invention belongs to the field of gene therapy, and more specifically relates to a recombinant promoter, carrier and application thereof for protein expression in the liver. A set of recombinant nucleic acid sequences for regulating the high expression of genes in the liver system disclosed by the invention. Compared with other sequences of similar size reported so far, the recombinant regulatory sequence fragment has significantly enhanced expression ability of driver reporter gene and human blood coagulation factor FVIII in the liver system, enhanced sustained high-level expression ability, and is suitable for recombinant adenovirus accompanying Viral (rAAV)-mediated gene therapy.

Description

technical field [0001] The invention belongs to the field of gene therapy, and more specifically relates to a recombinant promoter, carrier and application thereof for protein expression in the liver. Background technique [0002] Gene therapy refers to the introduction of exogenous normal genes into target cells to correct or compensate diseases caused by gene defects or abnormalities, and finally achieve the purpose of treatment. Since the 1990s, gene therapy technology has moved from laboratory research to clinical practice. From simple gene therapy for genetic diseases, it has rapidly expanded to the field of tumor treatment, and in recent years, it has been studied as an antiviral therapy and is being studied in depth. Liver gene therapy is to introduce therapeutic genes into the liver, use liver cells as biological response cells to express therapeutic genes, produce therapeutic proteins and enter the blood circulation to achieve therapeutic purposes; at the same time...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C07K14/755C12N15/864C12N15/65C12N5/10A01K67/027
CPCC12N15/113C07K14/755C12N15/86C12N15/65A01K67/0278C12N2750/14143C12N2800/107C12N2830/15C12N2830/008A01K2267/01A01K2227/105
Inventor 潘杏刘光猛张胜方文晶何晓斌
Owner JINFAN BIOMEDICAL TECH (WUHAN) CO LTD