Vector coexpressing dodecameric trail and suicide gene hsv-tk, and anticancer stem cell medicine using thereof

A technology of HSV-TK and suicide gene, which is applied in the field of anti-cancer stem cell therapeutic agents, can solve the problems of weak anti-cancer efficacy and difficulty in successful anti-cancer treatment, and achieve excellent anti-cancer effects

Inactive Publication Date: 2014-11-19
POSTECH ACAD IND FOUND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] TRAIL and HSV-TK have been used as safe candidate substances in targeted cancer therapy because they can induce cancer cell-specific apoptosis, and have been applied to the treatment of metastatic cancer by using mesenchymal stem cells, but only TRAIL or HSV-TK The anti-cancer effect of TK monotherapy is weak, and it is difficult to succeed in the clinical application of anti-cancer therapy (Loebinger et al., Cancer Res, 69:4134, 2009; Miletic et al. Molecular Ther, 15:1373, 2007)

Method used

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  • Vector coexpressing dodecameric trail and suicide gene hsv-tk, and anticancer stem cell medicine using thereof
  • Vector coexpressing dodecameric trail and suicide gene hsv-tk, and anticancer stem cell medicine using thereof
  • Vector coexpressing dodecameric trail and suicide gene hsv-tk, and anticancer stem cell medicine using thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1: Construction of tPA-SPD-TRAIL DNA cassette (dTRAIL)

[0069] The codon-optimized tPA secretion signal sequence and the codon-optimized SPD dodecamer formation sequence are chemically synthesized in a ligated form, the tPA secretion signal sequence has the nucleotide sequence of SEQ ID NO.4, the The SPD dodecamer forming sequence has the nucleotide sequence of SEQ ID NO.5. KpnI (5') and NotI (3') sites were added at its ends for efficient insertion into the vector. The tPA-SPD signal sequence was inserted into the pShuttle-Tet10 vector as an inducible adenovirus shuttle vector digested with KpnI and NotI to construct the pShuttle-Tet10 / tPA-SPD vector. pShuttle-Tet10, which is an inducible adenovirus shuttle vector, has CMV-Tet10 as an inducible promoter. Next, codon-optimized human TRAIL (114-281) having the nucleotide sequence of SEQ ID NO.6 was chemically synthesized. Also, NotI (5') and XbaI (3') sites were added to its ends for efficient insertion in...

Embodiment 2

[0070] Example 2: Construction of dTRAIL-IRES-TK cassette

[0071] Chemically synthesize the dodecamer human TRAIL sequence having the nucleotide sequence of SEQ ID NO.1 prepared in the above-mentioned embodiment 1 and codon optimization, the IRES sequence having the nucleotide sequence of SEQ ID NO.2, and Have the nucleotide sequence of SEQ ID NO.3 and the HSV-TK sequence after codon optimization, thereby construct DNA box ( figure 1 ). The constructed DNA cassette was inserted into the pShuttle vector as an adenovirus shuttle vector to construct the pShuttle / dTRAIL-IRES-TK vector. After digesting pShuttle / dTRAIL-IRES-TK with PmeI, homologous recombination was performed in the non-self-replicating pAd / Easy vector in BJ5183 competent cells. Afterwards, pAd / dTRAIL-IRES-TK containing the dTRAIL-IRES-TK cassette was screened using kanamycin antibiotic. For precise confirmation, it was digested with PacI to confirm a DNA band of 35+4.5kb. Then, pAd / dTRAIL-IRES-TK was transform...

Embodiment 3

[0072] Example 3: TRAIL and HSV-TK expression detection of MSC / dTRAIL-TK

[0073] Using the rAd / dTRAIL-IRES-TK prepared in the above-mentioned Example 2 and iron ions to transduce the bone marrow-derived mouse mesenchymal stem cells (rBM-MSC) for 30 minutes, to prepare the mesenchymal stem cells expressing the above-mentioned dTRAIL and HSV-TK simultaneously Mesenchymal Stem Cells (MSC) MSC / dTRAIL-TK. After 24 hours of transduction, the expression of human TRAIL was detected by Western blot and cell lysate, and the expression of HSV-TK was detected by RT-PCR ( figure 2 ). It was found that the prepared MSC / dTRAIL-TK expressed both TRAIL and HSV-TK.

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Abstract

The present invention relates to: a DNA cassette comprising a nucleic acid sequence coding for dodecameric TRAIL and a suicide gene nucleic acid sequence; a recombinant expression vector comprising the DNA cassette; a recombinant adenovirus made using the recombinant expression vector; a host cell transfected by means of the recombinant adenovirus; a composition for treating cancer comprising the host cell; and a cancer treatment method comprising a step of administering the composition for treating cancer to an individual. A stem cell therapeutic agent, with which dodecameric TRAIL and HSV-TK are simultaneously produced by introducing the DNA cassette of the present invention, has superior anticancer effects to those of existing therapeutic agents, and thus can effectively be used in the treatment of various solid cancers and metastatic cancers.

Description

technical field [0001] The invention relates to a carrier expressing dodecamer TRAIL (TNF-related apoptosis-inducing ligand) and HSV-TK (herpes simplex virus thymidine kinase) suicide gene simultaneously and an anti-cancer stem cell therapeutic agent using the same. In more detail, the present invention relates to a DNA cassette comprising a nucleotide sequence encoding dodecamer TRAIL and a suicide gene nucleotide sequence, a recombinant expression vector comprising the DNA cassette, and a recombinant expression vector prepared using the recombinant expression vector. An adenovirus, a host cell transfected with the recombinant adenovirus, a composition for cancer treatment comprising the host cell, and a method for treating cancer comprising the step of administering the composition for cancer treatment to an individual. Background technique [0002] Mesenchymal stem cells (MSCs) are pluripotent adult stem cells capable of differentiating into osteoblasts, chondrocytes, adi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/861C12N7/01A61K35/12
CPCC12Y207/01021C07K14/525A61K35/12C12N15/861A61K48/005C12N9/1211C12N15/86A61K35/50A61K35/28A61K38/00C12N2710/10043C12N2830/003C12N2800/22C07K2319/02C12N2710/10343A61P35/00C12N15/11C12N7/00A61K38/191A61K48/00
Inventor 成永喆金世原金秀珍朴常勋
Owner POSTECH ACAD IND FOUND
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