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Application of recombinant oncolytic poxvirus in preparation of pharmaceutical composition for treatment of lymphoma

A composition and lymphoma technology, applied in the fields of genetic engineering and oncology, can solve problems such as inability to obtain good curative effect, achieve enhanced killing ability, enhanced safety, and good anti-lymphoma effect

Active Publication Date: 2020-06-02
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these therapies have certain limitations, and often fail to achieve good curative effect. Therefore, it is of great significance to develop new safe and effective treatments to improve the survival of lymphoma patients

Method used

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  • Application of recombinant oncolytic poxvirus in preparation of pharmaceutical composition for treatment of lymphoma
  • Application of recombinant oncolytic poxvirus in preparation of pharmaceutical composition for treatment of lymphoma
  • Application of recombinant oncolytic poxvirus in preparation of pharmaceutical composition for treatment of lymphoma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Preparation of recombinant oncolytic vaccinia virus OVVscFvCD47

[0039] Follow the steps in order:

[0040] 1) Obtain the DNA sequence (SEQ ID NO.1) encoding scFvCD47 by gene synthesis method;

[0041] 2) Load the DNA sequence encoding scFvCD47 on the shuttle plasmid pCB to obtain pCB-scFvCD47 (SEQ ID NO.2), wherein the shuttle plasmid pCB was prepared according to the following literature: Fang Yourong, Li Hongyu, Chen Keda, Zhou Wen Shuo, Yan Hui. Construction of Zeocin and GFP dual selection marker recombinant vaccinia virus vector, International Journal of Epidemiology and Infectious Diseases, Volume 39, Issue 3, June 2012; it should be noted that other pCBs can also be used in the present invention , such as the shuttle plasmid pCB without a selectable marker.

[0042] 3), the plasmid pCB-scFvCD47 and wild-type vaccinia virus (purchased from ATCC, USA) were transferred into HEK293 cells, and in HEK293 cells (human kidney embryo cell line, purchased fr...

Embodiment 2

[0044] Example 2: Flow cytometry detection of recombinant oncolytic vaccinia virus carrying GFP (green fluorescent protein) respectively infected with human Raji, SUDHL-4 cell sources (respectively human Burkitt' lymphoma, diffuse large B-cell lymphoma cell line, The expression level of GFP was purchased from the Cell Bank of Chinese Academy of Sciences).

[0045] Inoculate 2×10 in a six-well plate 5 Each / mL Raji and SUDHL-4 cells were added with 0.1MOI, 0.5MOI, 1MOI, 5MOI and 10MOI of recombinant oncolytic vaccinia virus (OVVscFvCD47-GFP) carrying GFP, respectively, and the same amount of PBS was added to the control group, at 37°C , 5%CO 2 nourish. Cells were collected after 12h, 24h, and 48h, and the expression rate of GFP in each group of samples was analyzed by flow cytometry (BioRad). see results figure 1 , as the dose increased, the expression level of GFP increased, indicating that the recombinant oncolytic vaccinia virus OVVscFvCD47 can effectively invade lymphoma...

Embodiment 3

[0046] Example 3: Detection of the expression level of scFvCD47 in human Raji and SUDHL-4 cells infected by recombinant oncolytic vaccinia virus carrying the gene encoding scFvCD47 by PCR.

[0047] Recombinant oncolytic vaccinia virus (OVVscFvCD47) carrying scFvCD47 was used to infect Raji and SUDHL-4 cells at a dose of 4MOI at 37°C in 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 for reverse transcription into cDNA, and primer sequences (SEQ ID NO.3 and SEQ ID NO.4) were designed using Primer 5.0 software. The cDNA was subjected to fluorescent quantitative PCR by SYBR qPCR Mix, and the amplification system was 20 μL.

[0048] 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 AppliedBiosystems 7300real-time PCR instrument software. see results figure 2 , after treating Raji and SUDHL-4 cells wit...

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Abstract

The invention provides application of a recombinant oncolytic poxvirus in preparation of a pharmaceutical composition for treatment of lymphoma. An exogenous gene encoding scFvCD47 is inserted on an oncolytic poxvirus so as to obtain the recombinant oncolytic poxvirus (OVVscFvCD47), and the obtained recombinant oncolytic poxvirus is applied to the pharmaceutical composition for treatment of lymphoma. Gene therapy of malignant tumors is combined with viral therapy effectively, the oncolytic poxvirus capable of efficiently expressing scFvCD47 is prepared, and the ability of tumor killing is enhanced when the therapy is compared with pure gene therapy or viral therapy. A tumor-targeted therapy strategy is adopted, tumor cells can be targeted effectively, and specific proliferation in the tumor cells can be achieved, so that the safety of an oncolytic poxvirus vector is improved greatly. A virus replication mode for related gene deletion is adopted so as to ensure specific replication of the virus in tumor, and the safety of the oncolytic poxvirus vector is enhanced greatly. A good effect of lymphoma resistance is achieved through construction of the recombinant oncolytic poxvirus carrying the human scFvCD47 encoded sequence.

Description

technical field [0001] The invention belongs to genetic engineering and oncology, and relates to the use of a recombinant oncolytic vaccinia virus carrying the coding sequence of scFvCD47 in preparing a pharmaceutical composition, medicine or kit for treating lymphoma. Background technique [0002] Lymphoma is a group of malignant tumors originating from lymph nodes and other extranodal lymphoid tissues, with various types and high incidence. According to the "2018 Global Cancer Statistics", there are more than 18 million new cancer patients worldwide, and lymphoma accounts for about 3%. In my country, the total incidence of non-Hodgkin's lymphoma accounts for about 80% of the incidence of malignant lymphoma, and it is difficult to cure. Chemotherapy is still an important means of treatment of lymphoma. Although small-molecule chemical drugs have the advantages of good permeability and strong cytotoxicity, their specificity is low, and they often cause serious side effects...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/768A61K48/00A61P35/00
CPCA61K35/768A61K48/005A61P35/00
Inventor 钱文斌王世兵
Owner ZHEJIANG UNIV
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