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Recombinant oncolytic adenovirus expressing human interleukin 15 and construction method thereof

A technology of oncolytic adenovirus and human interleukin, which is applied in the field of new recombinant oncolytic adenovirus and its construction, can solve the problem that simple oncolytic virus is difficult to stimulate immune response, achieve enhanced anti-tumor effect, activate systemic immune response, improve The effect of anti-tumor effect of virus

Active Publication Date: 2015-12-23
晏阳
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, due to the existence of antiviral immunity in the body and the strong immunosuppressive network of tumors, it is difficult for simple oncolytic viruses to stimulate an ideal immune response.

Method used

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  • Recombinant oncolytic adenovirus expressing human interleukin 15 and construction method thereof
  • Recombinant oncolytic adenovirus expressing human interleukin 15 and construction method thereof
  • Recombinant oncolytic adenovirus expressing human interleukin 15 and construction method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Example 1 Recombinant oncolytic adenovirus expressing human interleukin-15 and its construction method

[0041] 1. Experimental materials and reagents

[0042] Plasmid pXC20 and adenovirus backbone plasmid pPE3-RC were provided by the Virus and Gene Therapy Laboratory of Shanghai Oriental Hepatobiliary Surgery Hospital. Plasmid hIL2SPIL15 carrying the hIL-15 gene. Restriction enzymes EcoRI, XhoI, SalI, PvuII, NcoI, etc. were purchased from NEB Company in the United States, DNA Ligase Solution I was purchased from TakaRa Company, gel recovery kit, PCR product recovery kit, plasmid DNA preparation kit, viral DNA The extraction kit was purchased from QIAGEN Company, and the LipofectAmine2000 kit was purchased from GIBCOBRL Company. Human embryonic kidney 293 cells (HEK293 cells) were purchased from MICROBIX BIOSYSTEMS, Canada. Human colon cancer cell line SW620 and human embryonic fibroblast cell line Wi38 were provided by the Institute of General Surgery, General Hospit...

Embodiment 2

[0083] Example 2 Effect of recombinant oncolytic adenovirus Ad-E2F1 / IL15 on cell proliferation

[0084] Human colon cancer cells SW620 and human embryonic lung fibroblasts Wi38 were plated into 96-well plates at an amount of 5000 per well, and after culturing for 6 h (cell attachment), Ad-E2F1 / IL15 virus (prepared in Example 1) and oncolytic virus Ad-E2F1 without IL15 gene, each group has 3 parallel wells, discards the culture medium containing the virus after the virus acts for 2 hours, and replaces it with 100 μL of 5% fetal bovine serum for cultivation liquid to continue culturing. On days 0, 1, 3, 5, and 7, take the corresponding 96-well plate, add MTT liquid (30 μL per well), incubate at 37°C for 4 hours, carefully suck out the culture supernatant in the well with a pipette, and add dimethyl 150 μL of sulfoxide solution was shaken by an oscillator for 20 minutes to fully melt the crystals, and a wavelength of 492 nm was selected to measure the light absorption value of ...

Embodiment 3

[0086] Example 3 Detection of IL-15 expression by ELISA after recombinant oncolytic adenovirus Ad-E2F1 / IL15 infected cells

[0087] Human colon cancer cells SW620 and human embryonic lung fibroblasts Wi38 in the logarithmic growth phase were inoculated into 6-well plates (1×10 5 / mL) was cultivated for 24h, adding the virus Ad-E2F1 / IL15 (prepared in Example 1) to the MOI as 5PFU / cell, and after 2h, replaced with 100 μL of 5% fetal bovine serum culture medium to continue culturing, and collected infected cells for 12, 24, and 48 hours. The cell culture supernatant was used to detect the expression level of IL-15 by ELISA.

[0088] 48h after Ad-E2F1 / IL15 infection, the IL-15 content in the culture supernatant of SW620 cells increased significantly, while the IL-15 in the supernatant of Wi38 cells remained at a low level ( Figure 11 ). It shows that Ad-E2F1 / IL15 can selectively lyse tumor cell SW620 while the expression of IL-15 is significantly increased, which meets the requ...

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Abstract

The invention provides a novel recombinant oncolytic adenovirus expressing human interleukin 15. Specifically, the gene promoter of a type 5 adenovirus E1 region is replaced with a transcription factor E2F-1 gene, and an hIL-15 gene is inserted to an E3 region to construct the recombinant oncolytic adenovirus. According to the invention, E2F-1 is used as the promoter to realize replication of virus specificity in tumor cells, and at the same time, loading of human IL-15 gene in a virogene E3 region can further enhance the anti-tumor effect of the virus. As the pRb / E2F pathway defects exist extensively in solid tumors, through the tumor resolving effect of the virus, a variety of tumor antigens from the individual itself can be acquired, and are not limited by antigen subcellular localization, thus being conducive to producing anti-tumor immune response with self tumor specificity, and having individualized and general treatment significance. In addition, the virus replication process drives the IL-15 gene expression, high concentration IL-15 can be obtained from a part of the virus-infected tumor cell, thereby being in favor of stimulating the activity of immune cells, activating the general immune response and strengthening the antitumor effect.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a novel recombinant oncolytic adenovirus expressing human interleukin-15 and its construction method. Background technique [0002] Malignant tumor is a major disease that threatens the health of our people. Traditional surgery, chemotherapy, and radiotherapy only reduce the tumor burden, but cannot cure tumor recurrence and metastasis. Since the occurrence and development of malignant tumors has its molecular genetic basis, gene therapy is expected to become a new option for tumor treatment. Among them, the genetic modification of adenovirus to target and kill tumor cells while avoiding the impact on normal cells (ie, oncolytic adenovirus) has become a new member of tumor gene therapy. [0003] In order to achieve tumor targeting of oncolytic viruses, in their construction strategy, tumor or tissue-specific promoters are often loaded in the viral genome to activate E1A, E1B ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N15/24A61K48/00A61K38/20A61P35/00
Inventor 晏阳杜晓辉徐迎新李荣
Owner 晏阳
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