Eureka AIR delivers breakthrough ideas for toughest innovation challenges, trusted by R&D personnel around the world.

Expression vector suitable for expressing coded sequence used for gene therapy

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

Active Publication Date: 2013-09-11
MOGAM BIOTECH RES INST
View PDF2 Cites 0 Cited by
  • 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

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Example 1: Isolation and activity analysis of liver tissue-specific expression promoters and enhancers

[0109] Isolation and activity analysis of liver tissue-specific expression promoter

[0110] Genomic DNA was extracted from cell lysates of a human hepatic cell line (Chang cells) using DNeasyTissueKit (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 DNA fragment having the nucleotide sequence of SEQ ID NO: 41. 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 was id...

Embodiment 2

[0133] Example 2: Isolation of hFIXUTR and introns of liver-specific genes, and analysis of gene expression efficiency of UTRs and introns

[0134] Identify the UTR of hFIX and construct the expression vector containing UTR

[0135] 1.2 mg of total RNA was extracted from 1 g of human liver tissue using an RNA extraction kit (PharmaciaBiotech), which was homogenized with a homogenizer in advance. Single-stranded DNA was synthesized using the extracted RNA as a template, M-MuLV reverse transcriptase (Takara), and oligo-dT17 primer (Takara), and double-stranded cDNA was synthesized from the single-stranded DNA using DNA polymerase I (Takara).

[0136]In order to evaluate the effect of FIXUTR on gene expression efficiency, PCR was carried out using cDNA as a template, a primer set (SEQ ID NO: 11 and 12) and Ex-Taq designed from the FIX nucleotide sequence (Genbank accession number NM000133) to obtain FIX cDNA of 'UTR (SEQ ID NO:62) and 3'UTR (SEQ ID NO:63). The PCR conditions a...

Embodiment 3

[0176] Example 3: Isolation of liver-specific LCR and analysis of gene expression efficiency of LCR

[0177] Isolate liver-specific LCR and construct a plasmid vector containing LCR

[0178] The position of the TGTTTGC motif was analyzed downstream of the α1-antitrypsin, alpha-fetoprotein and albumin genes expressed only in liver tissue. The results are shown in Figure 10 middle.

[0179] Such as Figure 10 As shown in , seven TGTTTGC motifs are distributed in -7.8kb of the α1-antitrypsin gene, six motifs are forward, and one motif is reverse; there is also a forward motif located in α1- The -108bp site of the antitrypsin gene. At the -3.8kb site of alpha-fetoprotein, two TGTTTGC motifs are distributed in the forward direction, and one motif is in the reverse direction; at the -6kb site of the albumin gene, one motif is in the reverse direction. In addition, TGTTTGC motifs clustered at the α1-antitrypsin gene-800bp site, the alpha-fetoprotein gene-1.5kb and -500bp sites...

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

Provided is an expression vector for gene therapy. The expression vector comprises a new combination which contains a transcriptional control element and comprises a promoter, an enhancer, an intron, an untranslated region (UTR) and a loca control region (LCP). The expression vector can continuously express specific genes of hepatic tissue so that the expression vector can be effectively used in treating thrombus forming, hemophilia, liver cancer and the like.

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 Containing son, untranslated region (UTR) and locus control region (locus control region, LCR), said vector can support 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...

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
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/85
Inventor 高垡经李圭铉李贤尹成泰赵义哲
Owner MOGAM BIOTECH RES INST
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
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