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Enhancer for promoter, and use thereof

A technology of promoter and intron, applied in the direction of introducing foreign genetic material using a vector, cells modified by introducing foreign genetic material, peptide source, etc., can solve the problem of poor cytomegalovirus activity and lack of intron enhancer function. fully understand the issues

Inactive Publication Date: 2012-09-26
NAT INST OF BIOMEDICAL INNOVATION HEALTH & NUTRITION +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the case of viral promoters such as herpesviruses, the enhancer function of introns is not well understood
[0005] The cytomegalovirus (CMV) promoter is known to be less active in lymphocytes
Although the enhancer of patent document 2 is used, it is unknown whether this activity increases in cells other than CHO cells

Method used

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  • Enhancer for promoter, and use thereof
  • Enhancer for promoter, and use thereof
  • Enhancer for promoter, and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0196] The major immediate early gene (MIE) promoter of human herpesvirus 6 (HHV-6) (6MIEp) was confirmed to be more active than the IE promoter of cytomegalovirus (CMV) in T cells (SEQ ID NO.35) activity. In this example, a promoter having an extended 3' end of the HHV-6B MIE promoter was prepared, and it was investigated to determine whether a similar effect could be obtained using the intron of IE1.

[0197] (Materials and methods)

[0198] The upstream region of the main immediate early gene of HHV-B strain (strain) HST is amplified by various primers described below ( figure 1 ).

[0199] Full Fw primer: 5'-TCTCTCGAGAGTTAAAGATCAGCGGGTAC-3' (SEQ ID NO.7);

[0200] - d1Fw primer: 5'-AGTCGGTACCGGCGAATGAGAACTCTAAAAGCTC-3' (SEQ ID NO.8);

[0201] - d2Fw primer: 5'-AGTCGGTACCTACTGTGGTTGGGGTCTTTCCTAC-3' (SEQ ID NO.9);

[0202] - d3Fw primer: 5'-AGTCGGTACCACATTCCTGTTTCATGATGTGTAGC-3' (SEQ ID NO.10);

[0203] - d4Fw primer: 5'-AGTCGGTACCTCCTGTTTTTGAGTAAGATATGAC-3' (SEQ ID NO...

Embodiment 2

[0218] (Examination of the addition reaction of polyadenylic acid obtained from herpes virus)

[0219] A polyA tail is added to the 3'-end of the mRNA. It is generally believed that the polyA tail can stabilize mRNA and promote protein expression. The polyA tail targets the sequence AAUAAA in the 3' region of the mRNA and is added thereto by polyA polymerase. Protein expression is attempted by inserting a region containing a polyA addition sequence obtained from a herpes virus downstream of a gene encoding a protein of interest.

[0220] (Materials and methods)

[0221] Regions shown below containing portions believed to be polyA addition sequences obtained from HHV-6B, U90, and U100 were amplified by PCR, respectively. These were inserted downstream of the DsRed2 gene in pBlueScriptSK(-)-DsRed2 vector (Clontech). Then, 6MIE was used as a promoter to construct plasmids pBleuScriptSK(-)-6MIE-DsRed2-U90pA (SEQ ID NO. 39) and pBlueScriptSK(-)-6MIE-DsRed2-U100pA (SEQ ID NO. 40...

Embodiment 3

[0228] (Example 3: Construction of specific deletion system)

[0229] Knockdown of gene expression in blood cells was performed using the IE promoter and the RNAi method together with the enhancer of the promoter of the present invention. Since the IE promoter is abundantly expressed in blood cells, the IE promoter is advantageous for analysis.

[0230] (1) Preparation of cells (in the case of macrophages)

[0231] Peripheral blood from healthy individuals was collected, separated and purified using the density gradient method using Ficoll / Hypaque. PBMCs were cultured in AIM V serum medium (Life Technologies) supplemented with M-CSF (R&D systems, 100 U / ml). The medium was changed every 3 days, and the macrophages on the 6th or 7th day after culture were used for the experiment.

[0232] (2) Production of siRNA expression retroviral vector

[0233] To express hairpin RNA, produce synthetic oligos containing a "sense strand target sequence", a "loop sequence", an "antisense ...

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Abstract

Disclosed is an enhancer for a viral promoter such as a promoter that can induce expression selectively and strongly in immunocompetent cells (e.g., lymphocytes) or blood cells. It is found unexpectedly that an intron has the above-mentioned enhancer activity. Thus, it is found that an enhancer for a promoter, which comprises an intron sequence for a major immediate early gene (MIE) of human herpes virus-6 (HHV-6) (particularly HHV-6B) or a fragment of the intron sequence, has a potent promoter activity.

Description

technical field [0001] The present invention relates to a promoter enhancer and its use. Background technique [0002] In order to treat various diseases targeting lymphocytes, such as leukemia and human immunodeficiency virus (HIV) infection, it has been required to establish a technique for gene therapy targeting lymphocytes. The present inventors have discovered the HHV-6B MIE promoter as a promoter for introducing a desired gene into lymphocytes from human herpesvirus-6 (HHV-6), a herpes virus, and filed their patent application (Patent File 1). [0003] This viral promoter can be used alone, but ideally is used in combination with an enhancer to enhance its effect. [0004] In this regard, in the case of eukaryotic promoters, various introns are known to function as enhancers. However, in the case of viral (eg, herpes virus) promoters, the enhancer function of introns is not well understood. [0005] The cytomegalovirus (CMV) promoter is known to be less active in l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N5/10
CPCC12N2710/16541C12N2740/10043C12N15/85A61K48/0066C12N2830/00C07K14/005C12N2830/60C12N2830/008C07K14/03C12N2710/16522C12N5/10C12N15/11
Inventor 松浦正明武本真清腰塚哲朗山西弘一森康子
Owner NAT INST OF BIOMEDICAL INNOVATION HEALTH & NUTRITION
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