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Expression vector suitable for expressing coded sequence for gene therapy

A technology of coding sequence and expression cassette, applied in the field of expression vector of gene therapy, can solve problems such as reduced activity

Active Publication Date: 2013-10-16
MOGAM BIOTECH RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When the 315bp fragment at the 5' end of the FVII promoter (501bp in size) was truncated, the liver-specific activity of the promoter was increased by about 30%, but when the 210bp fragment was truncated, the activity was reduced by about 20-30% (Pollak, W.S., etc. , J.Biol.Chem., 271(3):1738-1747(1996))

Method used

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  • Expression vector suitable for expressing coded sequence for gene therapy
  • Expression vector suitable for expressing coded sequence for gene therapy
  • Expression vector suitable for expressing coded sequence for gene therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Example 1 : Isolation and Activity Analysis of Liver Tissue-Specific Expression Promoters and Enhancers

[0112] Isolation and activity analysis of liver tissue-specific expression promoter

[0113]Genomic DNA was extracted from cell lysates of a human hepatic cell line (Chang cells) using the DNeasy Tissue Kit (Qiagen). To isolate the FVII promoter, PCR was performed using genomic DNA as a template, primer sets of SEQ ID NO: 1 and 2, and DNA polymerase (Ex-Taq, Takara) to obtain a nucleotide sequence having SEQ ID NO: 41 of DNA fragments. Specifically, PCR was carried out according to the following conditions: first denaturation at 94°C for 5 minutes; 30 cycles of denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds and extension at 72°C for 1 minute; final extension at 72°C for 3 minutes. The PCR product was purified by gel extraction and inserted into pCR2.1-TOPO plasmid vector. The FVII promoter having the nucleotide sequence of SEQ ID NO: 41 ...

Embodiment 2

[0137] Example 2: Isolation of hFIX UTRs and introns of liver-specific genes and analysis of gene expression efficiency of UTRs and introns

[0138] Identify the UTR of hFIX and construct an expression vector containing the UTR

[0139] 1.2 mg of total RNA was extracted from 1 g of human liver tissue previously homogenized with a homogenizer using an RNA extraction kit (Pharmacia Biotech). Single-stranded DNA was synthesized using the extracted RNA as template, M-MuLV reverse transcriptase (Takara) and oligo-dT17 primer (Takara), and double-stranded cDNA was synthesized from single-stranded DNA using DNA polymerase I (Takara).

[0140] To evaluate the effect of FIX UTR on gene expression efficiency, PCR was performed using cDNA as template, a primer set (SEQ ID NOs: 11 and 12) designed from the FIX nucleotide sequence (Genbank accession number NM000133) and Ex-Taq to FIX cDNA with 5'UTR (SEQ ID NO:62) and 3'UTR (SEQ ID NO:63) was obtained. PCR conditions were as follows: fi...

Embodiment 3

[0184] Example 3: Isolation of liver-specific LCRs and analysis of LCR gene expression efficiency

[0185] Isolation of liver-specific LCR and construction of plasmid vector containing LCR

[0186]The location of the TGTTTGC motif was analyzed downstream of the alpha1-antitrypsin, alpha-fetoprotein and albumin genes expressed only in liver tissue. Results are shown in Figure 10 middle.

[0187] like Figure 10 As shown in the figure, seven TGTTTGC motifs are distributed in the -7.8kb of the α1-antitrypsin gene, six motifs are forward, and one is reverse; and one forward motif is located in the α1- -108bp site of antitrypsin gene. At the -3.8kb site of alpha-fetoprotein, two TGTTTGC motifs are distributed in the forward direction, and one motif is reversed; at the -6kb site of the albumin gene, one motif is reversed. In addition, the TGTTTGC motif was clustered at the α1-antitrypsin gene-800bp site, the alpha-fetoprotein gene-1.5kb and-500bp site, and the albumin gene-10...

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Abstract

The invention provides an expression vector for gene therapy. The expression vector contains new combination of transcription regulation elements, and comprises a promoter, an enhancer, an intron, a UTR (untranslated region) and an LCR (locus control region). The expression vector can express specific genes of liver tissue continually, thus being applicable to treatment on thrombosis, hemophilia, liver cancer and the like effectively.

Description

[0001] This application is a divisional application of a Chinese patent application with application number 200880126705.0, and the original application is a Chinese national phase application of the international application PCT / KR2008 / 000870. technical field [0002] The present invention relates to an expression vector for gene therapy, more particularly to an expression vector for gene therapy comprising a new combination of transcriptional regulatory elements (cis-regulatory elements or trans-acting elements), including promoters, enhancers, internal Contains, untranslated region (UTR) and locus control region (locus control region, LCR), the vector can support the target gene to be expressed continuously at a high level in the liver. Background technique [0003] The functions of the liver include blood storage, glucose conversion, and secretion of various cytokines, as well as the expression of genes affecting many diseases such as genetic, cardiovascular, metabolic, h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/63
Inventor 高垡经李圭铉李贤尹成泰赵义哲
Owner MOGAM BIOTECH RES INST
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