Method for preparing novel oncolytic virus by exosome-like technology

A technology of oncolytic virus and exosomes, which is applied in the field of oncolytic virus preparation to achieve high targeting, improve efficacy, and reduce side effects

Pending Publication Date: 2021-12-24
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, exosome-mimetic technology is currently only used to wrap drugs for targeted delivery 33-35 , so far there has been no report on the use of its packaged virus
Moreover, it is widely believed that the technique is unlikely to be useful for encapsulating therapeutic entities as large as viruses

Method used

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  • Method for preparing novel oncolytic virus by exosome-like technology
  • Method for preparing novel oncolytic virus by exosome-like technology
  • Method for preparing novel oncolytic virus by exosome-like technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Infection efficiency of adenovirus Ad5 to cells with low CAR expression is low

[0056] CAR (the coxsackie and adenovirus receptor) is the main receptor of adenovirus Ad5 infected cells. Early studies have demonstrated that adenovirus Ad5 has low infection efficiency for cells with low CAR expression. 13,19 We found that the fluorescence intensity of 293T, A549, HCC-LM3 and Hepa1-6 cells stained with anti-CAR-PE was significantly increased compared with the isotype treatment group by flow cytometric analysis, indicating that 293T, A549, HCC-LM3 and Hepa1-6 High expression of CAR on the cell surface ( figure 1 a); while the surface of B16-F10, CT26.WT and H22 cells were stained with CAR antibody, there was no significant change in fluorescence intensity compared with the isotype treatment group; indicating that these cell lines do not express CAR; while K562 and Jurkat cells were stained with CAR antibody After that, compared with the isotype treatment group...

Embodiment 2

[0057] Example 2 Preparation of Exosome-mimetic Ad5 (EM / VSV-G Ad5) with VSV-G

[0058] Vesicular stomatitis Indiana virus G protein (VSV-G) is capable of mediating viral entry into all cell types tested so far and is widely used in gene transduction and gene therapy 26 . This study hopes to use Exosome-mimetic technology to take advantage of the broad tropism of VSV-G on cells to realize the retargeting of Ad5 and increase the infection efficiency of Ad5 on CAR low-expression cell lines.

[0059] First, we constructed 293T cells expressing VSV-G (293T-VSV-G), infected 293T-VSV-G cells with Ad5-GFP at an MOI of 5, cultured at 37°C for 72 hours, and collected the cells. The collected cells were divided into two parts, Part of the virus was purified by iodixanol density gradient centrifugation after freezing and thawing 3 times using the traditional protocol; Squeeze sequentially through polycarbonate membranes with pore sizes of 10 μm, 5 μm, and 1 μm, using 15%, 20%, and 40% i...

Embodiment 3

[0061] Example 3 EM / VSV-G Ad5-GFP can resist the effect of anti-Ad5 neutralizing antibody and the carried VSV-G mediates its entry into cells

[0062] To further prove that EM / VSV-G Ad5-GFP is encapsulated in vesicles, and that such encapsulation brings new properties, we used anti-Ad5 neutralizing antibody and VSV-G neutralizing serum for EM / VSV- G Ad5-GFP was processed for subsequent analysis. After treating EM / VSV-G Ad5-GFP and Ad5-GFP viruses with anti-Ad5 neutralizing antibodies at dilutions of 1 / 32, 1 / 64, and 1 / 128, the infection efficiency of Ad5-GFP virus was only that of the PBS-treated control group 5.66±2.1%, 9.3±2.02% and 12.6±3.84%, while the infection efficiency of EM / VSV-G Ad5-GFP was 61.0±3.10%, 71.3±3.7% and 81.6±1.2% ( figure 2 f) It can be seen that after treatment with anti-Ad5 neutralizing antibody, the Ad5-GFP virus almost lost its ability to infect, while the EM / VSV-G Ad5-GFP was infected by the exosome-like outer membrane and the presence of VSV-G. Ca...

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Abstract

The invention relates to the field of preparation of oncolytic viruses, in particular to a method for preparing a novel oncolytic virus by an exosome-like technology. The invention discloses the method for preparing the novel oncolytic virus with functions of targeting and escaping an antiviral neutralizing antibody by using the exosome-like technology. Specifically, after cells expressing a specific membrane protein, such as VSV-G, are infected with viruses and replicated, an exosome-like step-by-step extrusion technology is utilized to manufacture the oncolytic virus wrapped by the exosome-like, and the outer surface of the exosome-like retains the membrane protein on the cell surface, such as the VSV-G, in the preparation process. The novel virus prepared by the method can realize heavy targeting through the membrane protein, such as th VSV-G, infects a cell line which has corresponding protein on the surface and can be bound with the membrane protein, and increases the gene transduction efficiency of the virus, the neutralization effect of an antiviral antibody can be avoided due to the existence of the exosome-like membrane, and meanwhile, the preparation method also can increase the yield of the virus.

Description

technical field [0001] The present invention relates to the field of oncolytic virus preparation, in particular to the field of preparing novel oncolytic viruses by using exosome-like technology, and specifically relates to a method for preparing new oncolytic viruses using exosome-like technology. Background technique [0002] Many studies have proved that oncolytic viruses can break tumor immune tolerance and induce anti-tumor immunity, turning "cold" tumors into "hot" tumors 1 ; At the same time, oncolytic virus, especially adenovirus, can also be used as a gene therapy vector 2-5 , to express the gene product of interest locally in the tumor. Studies have shown that local expression of soluble PD-1, anti-PD-1 or anti-PD-L1 in OV-Ad5 tumors inhibits the PD-1 / PD-L1 pathway to induce anti-tumor immunity, significantly inhibits tumor growth, prolongs the survival of mice, and reduces Side effects of PD-1 / PD-L1 pathway blockade therapy 6,7 . This therapeutic strategy usin...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N15/85A61K48/00A61K38/17A61P35/00C12R1/93
CPCC12N7/00C12N15/85C07K14/005A61K48/0008A61K38/177A61P35/00C12N2710/10352C12N2710/16622
Inventor 吴俊华魏继武张永辉吴俊艺张海林刘淑雯马丁
Owner NANJING UNIV
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