Specific gene-virus therapeutic drug for colorectal cancer

A technology for colorectal cancer and therapeutic drugs, applied in gene therapy, medical raw materials derived from viruses/phages, drug combinations, etc., can solve problems such as non-targeting, and achieve high therapeutic effect.

Inactive Publication Date: 2013-04-24
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The gene carried by the oncolytic virus is OV-gene, but Ad-gene is g...

Method used

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  • Specific gene-virus therapeutic drug for colorectal cancer
  • Specific gene-virus therapeutic drug for colorectal cancer
  • Specific gene-virus therapeutic drug for colorectal cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Figure 1A : Schematic diagram of the construction principle of p Ad·(ST13)·CEA·E1A(Δ24)

[0040] Delete 24bp (deleted from 923-946bp) in the E1A of the adenovirus, and replace its natural promoter with the CEA promoter for regulation, and add a ST13 expression cassette (ST13) before CEA.

[0041] Figure 1B : Concrete steps of construction of Ad·(ST13)·CEA·E1A(Δ24) and its molecular structure diagram.

[0042] (1) Insert the CEA promoter into the plasmid pAd·E1A(Δ24) double-digested with XhoI and SnaB I to construct the plasmid pAd·CEA·E1A(Δ24);

[0043] (2) Digest pCA13-ST13 (commercially available) with EcoRV and HindIII, cut out the ST13 gene, and connect it to the pMD18-T Simple-HCMV-MCS-polyA (SV40) vector cut with the same enzyme to form pMD18- T Simple-HCMV-ST13-polyA(SV40) (Note: pMD18-T Simple is commercially available).

[0044] (3) Digest pMD 18-T Simple-HCMV-ST13-polyA(SV40) with SalI to obtain the "HCMV-ST13-polyA(SV40)" expression cassette; insert it ...

Embodiment 2

[0047] Example 2: Construction of Ad·CEA·E1A·E1B(Δ55)-(TRAIL-IETD-ST13) (Figure 2)

[0048] Figure 2A : The construction principle frame diagram of Ad·CEA·E1A·E1B(Δ55)-(TRAIL-IETD-ST13)

[0049] Figure 2B : The specific construction process of Ad·CEA·E1A·E1B(Δ55)-(TRAIL-IETD-ST13)

[0050] (1) pZD55 was cut with XhoI-SnaB1, and CEA with the same restriction site was loaded into it to obtain pZD55·CEA promoter (9234bp);

[0051] (2) The 9234bp plasmid was cut with XhoI-MfeI and joined with pSW (6568bp) digested with the same enzyme to obtain pSW·CEA·E1A·E1B(Δ55) (9033bp) (pSW was cut with XhoI-MfeI by the pShuttle of the existing commercial product Open, made by adding E1A·E1B);

[0052] (3) Cut pCA13·TRAIL-IETD-ST13 with BglII and load it into a 9033bp plasmid to obtain pSW·CEA·TRAIL-IETD-ST13, namely pAd·CEA·E1A·E1B(Δ55KD)-(TRAIL-IETD-ST13 )(11582bp) (Δ in the first bracket means deletion of 55KDa gene, non-expression box, the last bracket is expression box);

[0053]...

Embodiment 3

[0056] Example 3: In vitro (invitro) anticancer effect of Ad·(ST13)·CEA·E1A (Δ24) ( Figure 3A and Figure 3B )

[0057] Figure 3A : Dose-dependent in vitro anticancer effect

[0058] ONYX-015, Ad·(EGFP)·CEA·E1A (Δ24) and Ad·(ST13)·CEA·E1A (Δ24) were infected with the following different doses of virus (MO1 0.1, 1.0, 5.0), respectively.

[0059] Four days later, it was detected by MTT method, and the result was the mean ± SD of three detections.

[0060] The result is given by Figure 3A It can be seen that anticancer drugs have greater killing effects on different colorectal cancer cells SW620, HT116, and HT29 ( Figure 3A ), while all drugs had no killing effect on normal cell groups QSG7701 and WI38 ( Figure 3A ), the killing effect on Hela is less than that of colorectal cancer.

[0061] Figure 3B : Time-dependent in vitro anticancer effect

[0062] Inoculate cancer cells and normal cells in 24-well plates (the number of cells is 5-10×10 3 ), when the cells gr...

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Abstract

The invention discloses a specific gene-virus therapeutic drug for colorectal cancer. A natural promoter of an early gene E1A of an adenovirus is substituted by a colorectal cancer specific promoter CEA to form an oncolytic adenovirus carrying a specific antitumor gene ST13 of colorectal cancer. If the antitumor effect of the oncolytic adenovirus is not strong enough to kill all the cancer cells, the following genes may be added to a same vector with the oncolytic adenovirus for common usage: TRAIL, IL-24, MnSOD, CD, Smac, GM-CSF, IFN, IL-12, p53, RNAi, microRNA, Caspase 3 and Ba*; or two genes can be connected through various schemes. The gene-virus therapeutic drug for colorectal cancer provided by the invention has high therapeutic effect on targeting colorectal cancer, and has no effect on normal cells.

Description

technical field [0001] The invention belongs to the field of Cancer Targeting Gene-Viro-Therapy (CTGVT), in particular to Cancer Targeting Gene-Viro-Therapy Specific for Colorectal Cancer. CTGVT-CRC), and more specifically, a colorectal cancer-specific gene-viral therapy. Background technique [0002] From 1999 to 2001, Liu Xinyuan created a cancer treatment strategy called Cancer Targeting Gene-Viro-Thearpy (CTGVT), which is to insert anti-cancer genes into oncolytic virus , OV), so CTGVT strategy is OV-(gene) strategy or Gene Armed Oncolytic Virus Therapy (GAOVT) strategy, the latter is also OV-(gene), CTGVT (GAOVT) combines the advantages of gene therapy and oncolytic virus therapy Combined, because the oncolytic virus itself has an anti-cancer effect, it may specifically replicate hundreds of times in cancer cells, and the anti-cancer gene inserted in it can also replicate hundreds of times, so its anti-cancer effect is greatly improved. It is much better than the corr...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K35/76A61P35/00
Inventor 刘新垣杨敏周秀梅谢国良
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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