Multi-gene fusion oncolytic adenovirus and construction method and application thereof

An oncolytic adenovirus and multi-gene technology, which is applied in the field of multi-gene fusion oncolytic adenovirus and its construction, can solve the problem that the efficiency of tumor-specific promoters cannot meet expectations, and can improve the tumor immune microenvironment and activate the body. The effect of anti-tumor immune response, promoting tumor cell apoptosis, and improving the level of treatment

Pending Publication Date: 2021-11-26
MEI HOSPITAL UNIV OF CHINESE ACAD OF SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in practice, the curative effect of oncolytic adenoviruses is often restricted by many factors, such as the ubiquitous presence of type 5 adenovirus antibodies in the human body, which can clear oncolytic oncolytic adenoviruses; the efficiency of tumor-specific promoters that initiate viral replication; less than expected

Method used

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  • Multi-gene fusion oncolytic adenovirus and construction method and application thereof
  • Multi-gene fusion oncolytic adenovirus and construction method and application thereof
  • Multi-gene fusion oncolytic adenovirus and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Construction and preparation of recombinant adenovirus rAd.DCN.CD40L

[0054] The method is to synthesize DCN gene (decorin gene) and CD40L gene, insert DCN into pshuttle-cmv vector to obtain pShuttle-cmv-DCN (pSh.cmv.DCN); Viral E1A gene, natural E1B promoter, CD40L gene, internal ribosomal entry site (IRES) and E1B55K are connected in sequence, cloned into TE vector to obtain TE-TP-E1A-CD40L, and digested with restriction endonuclease MfeI TE-TP-E1A-CD40L, obtain the replication-related genes, clone them into the corresponding site of pSh.cmv.DCN, and obtain the shuttle plasmid pSh.cmv.DCN.CD40L.RE; refer to the Adeasy system to obtain the recombinant adenovirus plasmid pAd .DCN.CD40L.RE; and the oncolytic adenovirus rAd.DCN.CD40L was prepared in HEK293 cells. Specifically:

[0055] (1) Construction of pSh.cmv.DCN

[0056] Synthesize the full sequence of the DCN gene (as shown in Table 1) by gene synthesis technology, and add SalI and XhoI enzyme cutting ...

Embodiment 2

[0070] Example 2 In vitro function verification of oncolytic adenovirus rAd.DCN.CD40L

[0071] The method is, in colorectal cancer cells, using sulforhodamine B (SRB) staining method to detect the killing effect of oncolytic virus; through fluorescent quantitative detection of target genes DCN and CD40L in colorectal cancer cells Express. Specifically:

[0072] (1) Detection of killing ability of oncolytic adenovirus rAd.DCN.CD40L

[0073] Human colorectal cancer cell lines RKO, HCT116 and mouse colorectal cancer cell line CT26 were divided into 1×10 3Cells / well were seeded in 96-well plates. The next day, the oncolytic adenovirus rAd.DCN.CD40L and the control virus rAd.Null, rAd.DCN, rAd.CD40L and Ad.Null were respectively infected with 5-fold serial dilutions (1.25×10 6 Vps / cell-80Vps / cell). On the seventh day (sixth day after infection), SRB staining was used to detect the survival rate of the cells in each well, so as to clarify the cell killing effect of the oncolyti...

Embodiment 3

[0076] Example 3 Evaluation of curative effect of oncolytic adenovirus rAd.DCN.CD40L on colorectal cancer xenografts in mice

[0077] The method is to use mouse colorectal cancer cell CT26 to establish a transplanted tumor model, administer oncolytic virus intratumorally for treatment, and monitor its growth to evaluate the effect of oncolytic virus on mouse colorectal cancer transplanted tumors. Therapeutic effect. Specifically:

[0078] Single cell suspension of mouse colorectal cancer cells in 2×10 6 Cells / mouse, Babl / c mice subcutaneously bearing tumors for 6-8 weeks, observe the growth of tumors, detect the volume of tumors 7 days after tumor bearing, and randomly divide them into Buffer group and rAd. Null group, rAd.DCN, rAd.CD40L and rAd.DCN.CD40L groups. According to 2.5×10 10 VPs / 100μl dose, intratumoral injection of recombinant adenovirus for treatment. After treatment, the health status of the mice and the growth volume of the tumor were observed. The experim...

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Abstract

The invention discloses a multi-gene fusion oncolytic adenovirus. The oncolytic adenovirus is based on an Adeasy vector system and is inserted into a DCN gene expression cassette, an adenovirus replication regulation gene sequence and a CD40L gene expression cassette. The oncolytic adenovirus is used as a vector, and combined with treatment strategies of targeting TGF beta and targeting CD40-CD40L, the anti-tumor immune activation effect of the oncolytic adenovirus is synergistically exerted, and the treatment level of colorectal cancer is improved. After local administration of a tumor, tumor cells are killed through the oncolytic effect; and meanwhile, the continuously replicated virus can continuously generate a large amount of target proteins decorin and CD40L. The local high-concentration decorin can block abnormally activated TGF beta signals in the tumor cells and inhibit expression and secretion of transfer-related proteins. Meanwhile, the CD40L can activate the anti-tumor immune response of the body, inhibit tumor cell proliferation and promote tumor cell apoptosis. The effects of the oncolytic adenovirus in improving the tumor immune microenvironment and activating the anti-tumor immune response of the body are synergistically exerted, and the effect of the oncolytic virus is exerted more long-acting.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a multi-gene fusion oncolytic adenovirus and its construction method and application. Background technique [0002] In recent years, the incidence of colorectal cancer in my country has been increasing year by year, ranking second only to lung cancer. At present, the main treatment for colorectal cancer is surgical resection, and the effects of radiotherapy and chemotherapy are not satisfactory. Therefore, it is urgent to explore a more effective treatment method. [0003] In recent years, oncolytic virus therapy has developed rapidly, and a number of oncolytic virus therapeutic drugs have entered the phase II-III clinical research phase. In October 2015, the US FDA approved herpes simplex oncolytic disease (T-VEC) carrying granulocyte-macrophage colony stimulating factor (GM-CSF) to enter the market for the treatment of oncolytic virus. Injected a booster. At present...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/85C12N15/12A61K35/761A61K48/00A61K38/17A61P35/00C12R1/93
CPCC12N7/00C12N15/85C07K14/70575C07K14/4725A61K35/761A61K48/005A61K38/1709A61K38/177A61P35/00C12N2710/10321C12N2710/10352C12N2710/10332C12N2800/107
Inventor 杨月峰荣晔婧蔡挺张顺张沛瑶
Owner MEI HOSPITAL UNIV OF CHINESE ACAD OF SCI
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