Application of recombinant oncolytic virus in preparation of drugs for treating digestive tract cancer

A digestive tract cancer and oncolytic technology, applied in the fields of genetic engineering and oncology, can solve the problems of low expression level of IL-33 and poor prognosis, and achieve the effect of better resistance to gastric cancer, enhanced safety, and enhanced lethality

Pending Publication Date: 2020-05-15
杭州荣谷生物科技有限公司
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

In addition, IL-33 protein levels in lung adenocarcinoma patients were negatively correlated with tumor grade and size, suggesting that low IL-33 expression levels are associated with poor prognosis

Method used

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  • Application of recombinant oncolytic virus in preparation of drugs for treating digestive tract cancer
  • Application of recombinant oncolytic virus in preparation of drugs for treating digestive tract cancer
  • Application of recombinant oncolytic virus in preparation of drugs for treating digestive tract cancer

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Experimental program
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Embodiment 1

[0040] Embodiment 1, the preparation method of recombinant oncolytic vaccinia virus, the following steps are carried out in sequence:

[0041] 1) Obtain the DNA sequence (SEQ ID NO.1) encoding IL-33 by gene synthesis method;

[0042] 2) Load the sequence encoding IL-33 on the shuttle plasmid pCB to obtain pCB-IL-33 (SEQ ID NO.2), wherein the shuttle plasmid pCB was prepared according to the following literature: Fang Yourong, Li Hongyu, Chen Koda, Zhou Wenshuo, Yan Hui. Construction of Zeocin and GFP double selection marker recombinant vaccinia virus vector, International Journal of Epidemiology and Infectious Diseases, Volume 39, No. 3, June 2012; it should be noted that the present invention also Other pCBs can be used, such as the shuttle plasmid pCB without a selectable marker.

[0043] 3), the plasmid pCB-IL-33 and wild-type vaccinia virus (purchased from ATCC, USA) were transferred into HEK293 cells, and in HEK293 cells (human embryonic kidney cell line, purchased from ...

Embodiment 2

[0045] Embodiment 2: Flow cytometry detects that recombinant oncolytic vaccinia virus OVV-IL-33-GFP carrying GFP (green fluorescent protein) infects human HGC-27, SW-480, ASPC-1 cells (respectively human gastric cancer, colon GFP expression levels in cancer, pancreatic cancer cell lines).

[0046] Inoculate 2×10 in a six-well plate 5 HGC-27, SW-480, and ASPC-1 cells / mL (purchased from the Cell Bank of the Chinese Academy of Sciences), were added with 0.1 MOI, 0.5 MOI, 1MOI, 5MOI, and 10 MOI of recombinant oncolytic vaccinia virus carrying GFP (OVV-IL-33-GFP), the same amount of PBS was added to the control group, 37 ° C, 5% CO 2 to cultivate. Cells were collected after 24 hours, and the expression rate of GFP in each group of samples was analyzed by flow cytometry (BioRad). see results figure 1 , as the dose of OVV-IL-33 increased, the expression level of GFP increased, indicating that the recombinant oncolytic vaccinia virus can effectively invade gastric cancer, colon ca...

Embodiment 3

[0047] Example 3: PCR detection of the expression level of human cytokine IL-33 in human HGC-27, SW-480, ASPC-1 cells infected by recombinant oncolytic vaccinia virus carrying the gene encoding human cytokine IL-33.

[0048] Recombinant oncolytic vaccinia virus (OVV-IL-33) carrying the human cytokine IL-33 coding gene was used to infect HGC-27, SW-480, ASPC-1 cells at a dose of 4 MOI, 37 ° C, 5% CO 2 Conditioned for 24 hours. The total cellular RNA was extracted according to the TRIZOL method, and the extracted total RNA was used as a template to reverse transcribe into cDNA, and Primer 5.0 software was used to design primers (SEQ ID NO: 3). The cDNA was subjected to fluorescent quantitative PCR by SYBR qPCR Mix, and the amplification system was 20 μL.

[0049] The amplification parameters were: 95°C for 1 min, 95°C for 15 s, 60°C for 1 min, 40 cycles. The results were analyzed by AppliedBiosystems7300 real-time PCR instrument software. see results figure 2 , after OVV-IL...

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Abstract

The invention provides an application of a recombinant oncolytic virus in the preparation of drugs for treating digestive tract cancer, belongs to genetic engineering and oncology, and relates an application of a human IL-33-carrying recombinant oncolytic vaccinia virus in treating cancer. Through the effective combination of gene therapy for malignant tumors and viral therapy, an oncolytic vaccinia virus capable of efficiently expressing IL-33 can be prepared; and compared with simple gene therapy or viral therapy, killing abilities on tumors can be enhanced; through the adopting of a tumor targeted therapy strategy, tumor cells can be effectively targeted, and specific proliferation can be achieved, so that the safety of an oncolytic vaccinia virus vector can be greatly enhanced; throughthe adopting of the mode of virus replication related gene deletion, the intratumoral specific replication of the virus can be ensured, and the safety of the oncolytic vaccinia virus vector can be greatly enhanced; and through the construction of the human IL-33 carried recombinant oncolytic vaccinia virus, good anti-gastric, colon and pancreatic cancer effects can be achieved.

Description

technical field [0001] The invention belongs to genetic engineering and oncology, and relates to the use of a recombinant oncolytic vaccinia virus carrying an IL-33 coding gene in preparing a pharmaceutical composition, medicine or kit for treating gastric cancer, colon cancer and pancreatic cancer. Background technique [0002] According to the "2018 Global Cancer Statistics", there are more than 18 million new cancer patients worldwide. Among them, the new patients with gastric cancer, colon cancer and pancreatic cancer were 6.1%, 5.7% and 2.5% respectively, while the mortality rates were 5.8%, 8.2% and 4.5% respectively. High morbidity and high mortality are important characteristics of gastrointestinal cancer, and it is of great significance to develop new safe and effective treatment methods to improve the survival of patients with gastrointestinal cancer. [0003] Oncolytic virus immunotherapy (Oncolytic Virotherapy) is a new method of tumor treatment that has develop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/768A61K38/20A61P35/00
CPCA61K35/768A61K38/20A61P35/00A61K2300/00
Inventor 钱文斌王世兵王俊杰黄筛
Owner 杭州荣谷生物科技有限公司
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