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Conditionally replicating adenovirus expressing REIC gene

An adenovirus, replicating technology, applied in the direction of virus, virus/bacteriophage, double-stranded DNA virus, etc., can solve the problems of disease progression, weakened anti-cancer immune function, and limit the therapeutic effect of T-VEC, and achieve a strong anti-cancer effect , the effect of strong therapeutic effect

Active Publication Date: 2017-02-22
UNIV OKAYAMA +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cytokine GM-CSF has also been reported to induce immune activation at high doses, in addition to the activation of anticancer immunity by inducing the differentiation of dendritic cells as cancer antigen-presenting cells (Non-Patent Document 1). Inhibition of lineage cells weakens the anti-cancer immune function and worsens the disease (Non-Patent Document 4), which may limit the therapeutic effect of T-VEC

Method used

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  • Conditionally replicating adenovirus expressing REIC gene
  • Conditionally replicating adenovirus expressing REIC gene
  • Conditionally replicating adenovirus expressing REIC gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Example 1 Production of Oncolytic Adenovirus (Oncolytic Adenovirus: Oncolytic Ad)

[0113] In order to make oncolytic adenovirus expressing REIC or REIC C domain (REIC-C) and decorin (DCN) in the E1 region and E3 region respectively, pCA14Ad E1 shuttle vector expressing REIC or REIC C was firstly made. The REIC or REIC C gene was excised from pShuttole / REIC or REIC-C using NheI-blunt-HindIII and subcloned into pCA14Ad E1 shuttle vector previously digested with XbaI-blunt-HindIII. The pCA14 / REIC and pCA14 / REIC-C vectors were digested with BglII, and then the CMV-REIC-polA and AMV-REIC-C-polA expression cassettes were cloned into the pΔE1sp1B-HmT-Rd19 shuttle vector (Kim E et al., Hum Gene Ther 2003; 14:1415-1428; Kim JH et al., J Natl Cancer Inst2006; 98:1482-1493), pΔE1sp1B-HmT-Rd19 / REIC and pΔE1sp1B-HmT-Rd19 / REIC-C adenovirus E1 shuttle vector. The pSP72-E3 / DCN (I-KChoi et al., Gene Therapy 2010; 17:190-201.) E3 shuttle vector was linearized with XmnI and co-transfo...

Embodiment 2

[0116] Example 2 Expression of REIC protein in various cells caused by addition of oncolytic Ad, oncolytic Ad-REIC and oncolytic Ad-REIC domain

[0117] method

[0118] To measure REIC protein expression after Ad-REIC treatment, cells were seeded into flat-bottom 6-well plates and incubated for 24 hours. Using the MOI (multiplicity of infection, multiplicity of infection) recorded in the figure, the adenovirus was used to infect the cells in complete medium (300 μl) for 1 hour, and washed twice with PBS (phosphate buffered saline, phosphate buffered saline). Use lysis buffer (50 mM HEPES, pH 7.4, 250 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM DTT, 1 mM PMSF, 5 μg / ml leupeptin, 5 μg / ml aprotinin, 2 mM Na 3 VO 4 , 1mM NaF, 10mMβ-GP) were dissolved to extract the REIC protein.

[0119] After centrifugation, the supernatant was diluted with the same volume of 4×SDS sample buffer and heated at 95°C for 5 minutes. The samples (5 μg of protein) were separated on a 10% SDS-PAGE gel, and e...

Embodiment 3

[0123] Example 3 Confirmation of cell death induction rate in various cells by addition of oncolytic Ad and oncolytic Ad-REIC

[0124] method

[0125] To study the cytocidal rate after Ad-REIC treatment, cells were seeded into flat-bottom 6-well plates and incubated for 24 hours. The cells were treated with adenovirus in complete medium (300 μl) at the MOI (multiplicity of infection) described in the figure for 1 hour, and 1700 μl of fresh medium was added. After the number of days indicated in the figure, the dead cell rate (%) was determined by microscopic observation of 5 fields. It should be noted that, in Figure 4~6 In, data are presented as mean ± standard deviation. Tests for statistically significant differences were performed using scatter analysis or the Mann-Whitney U test. When p<0.05, it was judged to have a significant difference.

[0126] result

[0127] The cell death induction rate in various cells caused by the addition of oncolytic Ad and oncolytic Ad...

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Abstract

The purpose is to provide a conditionally replicating adenovirus having a strong anticancer effect. A conditionally replicating adenovirus growing specifically in cancer cells and expressing an REIC protein or REIC C domain protein, having full-length REIC DNA or REIC C domain DNA inserted into a conditionally replicating adenovirus including an (inverted terminal repeat) ITR sequence of a type 5 adenovirus and having an HRE sequence, hTERT promoter, DNA encoding decorin, and DNA encoding a peptide including an RGD sequence inserted.

Description

technical field [0001] The invention relates to a conditional replication adenovirus highly expressing REIC (REIC / Dkk-3) protein. Background technique [0002] So far, clinical trials of drugs against cancer using conditionally replicative (genetically engineered to proliferate specifically in cancer cells) various viruses have been reported (Non-Patent Document 1). Some results have been obtained in the treatment of cancer using these drugs, but on the other hand, the effects are often limited, and development of more effective drugs is desired. [0003] Representative examples of cancer-targeting drugs using conditional replicating viruses for which clinical trial results have been reported so far include Telomelysin (Non-Patent Document 2), which uses adenovirus type 5 as a backbone, and Talimogene, which uses herpes simplex virus type 1 as a backbone. laherparepvec (T-VEC, formerly known as Oncovex) (Non-Patent Document 3). [0004] In recent years, as shown in Non-Pat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01A61K35/761A61P35/00A61P37/04C12R1/93
CPCC12N15/86C12N2710/10343C12N2710/10332A61K35/761A61P35/00C12N2820/007C07K14/4703C07K14/4747C12N7/00C12N15/09A61K35/76C12N2710/10042C12N2710/10033
Inventor 公文裕巳那须保友渡部昌实尹彩钰
Owner UNIV OKAYAMA
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