Method for preparing novel oncolytic virus EM/VSV-G Ad5sPVRCD137L

A VSV-G, 293T-VSV-G technology, applied in the field of oncolytic virus preparation, can solve the problem of low natural exosome production

Pending Publication Date: 2021-05-28
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Artificial vesicle technology can overcome the problem of low yield of natural exosomes. At present, artificial vesicle technology is only used to encapsulate drugs to achieve targeted delivery. So far, there has been no report on the use of its encapsulated 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 EM/VSV-G Ad5sPVRCD137L
  • Method for preparing novel oncolytic virus EM/VSV-G Ad5sPVRCD137L
  • Method for preparing novel oncolytic virus EM/VSV-G Ad5sPVRCD137L

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Infection efficiency of adenovirus Ad5 to cells with low CAR expression is low

[0070] 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

[0072] Example 2 Vesicle-like technology used to prepare novel oncolytic virus EM / VSV-GAd5sPVRCD137L with VSV-G

[0073] 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 vesicle-like 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 cell lines with low CAR expression.

[0074] First, we constructed 293T cells expressing VSV-G (293T-VSV-G), infected 293T-VSV-G cells with Ad5sPVRCD137L at an MOI of 5, and collected cells after culturing at 37°C for 72 hours. The collected cells were divided into two parts, and one part was used After freezing and thawing 3 times in the traditional protocol, the virus was purified by density gradient centrifugation with iodixanol; the other part produced a new oncolytic virus EM / VSV-G Ad5sP...

Embodiment 3

[0080] Example 3 EM / VSV-G Ad5sPVRCD137L prepared by EM / VSV-G technology has higher infection efficiency, ability to express and secrete soluble PVRCD137L and oncolytic ability in vitro

[0081] The oncolytic adenovirus Ad5sPVRCD137L is an oncolytic adenovirus Ad5 expressing soluble PVRCD137L (named Ad5sPVRCD137L), and the genome structure of the oncolytic adenovirus Ad5sPVRCD137L is shown in image 3 shown. The low infection rate of oncolytic adenovirus Ad5sPVRCD137L to tumor types with low CAR expression severely limits the transformational application of Ad5sPVRCD137L. We applied EM / VSV-G technology to oncolytic adenovirus Ad5sPVRCD137L, expecting to significantly increase the infection efficiency of CAR low-expressing tumor cell lines through EM / VSV-G technology, and improve the therapeutic effect and expansion of oncolytic adenovirus Ad5sPVRCD137L its scope of application. The virus prepared by the oncolytic adenovirus Ad5sPVRCD137L through EM / VSV-G technology is named E...

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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 through a vesicle-like technology. The invention discloses a novel method which comprises the following steps: infecting cells capable of expressing specific membrane protein VSV-G with a virus Ad5sPVRCD137L, enabling the cells to pass through membrane holes by utilizing a centrifugal extrusion / shearing method so as to form vesicles, preparing the oncolytic virus wrapped by the vesicles, and reserving the membrane protein VSV-G on the cell surfaces on the outer surfaces of the vesicles in the preparation process. The prepared novel virus can realize heavy targeting through the membrane protein VSV-G, the gene transduction efficiency of the virus is increased, the neutralizing effect of an antiviral antibody can be avoided, the yield of the virus can be increased, and the soluble fusion protein sPVRCD137L expressed by the virus can play the dual functions of blocking an immune detection point and activating immune costimulation.

Description

technical field [0001] The invention relates to the field of preparation of oncolytic viruses, in particular to a method for preparing novel oncolytic virus EM / VSV-G Ad5sPVRCD137L by centrifugal extrusion / shearing production of vesicle-like technology. Background technique [0002] Cancer is one of the most harmful diseases to human life and health, and millions of people die of cancer every year. Many patients are already in the middle and late stages at the time of diagnosis and thus lose the opportunity for surgical treatment. Traditional radiotherapy and chemotherapy have not brought breakthrough progress to the tumor. Even small molecule targeted drugs are facing a huge challenge of short-term recurrence . [0003] In the past ten years, exciting results have emerged in the clinical research of anti-tumor immunotherapy, bringing hope to cancer patients again. Simultaneously blocking immune negative regulation and activating co-stimulatory pathways is proven to be an e...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N5/10C12N15/861C12N15/47C12R1/93
CPCC12N7/00C12N15/86C07K14/005C07K14/70575C07K14/70596C12N2760/20222C12N2710/10021C12N2710/10043C12N2710/10032
Inventor 吴俊华魏继武张海林张永辉吴俊艺刘淑雯马丁
Owner NANJING UNIV
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