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Application of attenuated yellow disease virus in oncolysis

A yellow fever virus and virus technology, which is applied in the field of biomedicine and achieves the effect of wide application prospect and small toxic and side effects

Pending Publication Date: 2021-11-30
BEIJING SHUNLEI BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that the yellow fever vaccine YFV-17D replicates and kills mouse and human tumor cell lines, but not untransformed human cells

Method used

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  • Application of attenuated yellow disease virus in oncolysis
  • Application of attenuated yellow disease virus in oncolysis
  • Application of attenuated yellow disease virus in oncolysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Embodiment 1: Construction and virus rescue of flavivirus attenuated strain clone

[0112] 1. Cloning construction of flavivirus attenuated strain

[0113] Schematic diagram of the cloning construction of attenuated flavivirus strains figure 1 shown.

[0114] Using the plasmid DNA of the infectious clone of the wild-type (WT) West Nile virus (WNV) strain as the backbone, the sequence from SLI to CS2 (including the 5' Terminal stem-loop region I (SLI), stem-loop region II (SLII), repeat circularization sequence region 3 (RCS3), stem-loop region III (SLIII), stem-loop region IV (SLIV), circularization sequence region 3 (CS3 ), dumbbell region 1 (DB1), repeat circularizing sequence region 2 (RCS2), dumbbell region (DB2), circularizing sequence region 2 (CS2)) deletion, retaining circularizing sequence region 1 (CS1) in the 3' untranslated region ) and the short hairpin-3' stem-loop region (sHP-3'SL) after circularizing sequence region 1, and inserting a poly(A) sequence...

Embodiment 2

[0131] Example 2: Oncolytic effect of WNV-poly(A) on tumor cells in vitro

[0132] In this embodiment, after infecting tumor cells with the WNV-poly(A) virus attenuated virus (WNV-poly(A)) prepared in Example 1 with a fixed MOI dose, observe its killing effect on tumor cells in bright field ( figure 2 A), and CCK8 was used to detect the killing of tumor cells by different doses of WNV-poly(A) to determine the oncolytic effect of the virus in vitro ( figure 2 B). in:

[0133] Control group (MOCK): tumor cell group cultured in medium without virus;

[0134] Experimental group: the tumor cell group cultured in the medium added with WNV-poly(A).

[0135] 1. Bright field observation of the oncolytic effect of WNV-poly(A) on tumor cells and normal cells

[0136] 12 kinds of tumor cells Huh7, A549, PC9, H1975, SKOV3, U87MG, U118MG, B16F1, 4T1, HELA, A375 and A2058 and 3 kinds of normal cells MLE-12, MEF and L-02 were divided into 8×10 4 Cells / well were seeded in 12-well cell c...

Embodiment 3

[0142] Example 3: The killing difference of WNV-wT and WNV-poly(A) on normal cells and tumor cells in vitro

[0143] This embodiment is obtained by transfecting the wild-type West Nile virus (WNV-WT by WNV infectious clone 3356 strain) and the WNV-poly (A) virus (WNV-poly) virus prepared in Example 1 with a fixed MOI dose. (A) After infecting tumor cells, its killing effect on normal cells and tumor cells was observed in bright field ( image 3 A), and use CCK8 to detect the killing of different doses of WNV-WT and WNV-poly(A) to normal cells and tumor cells to determine the difference in killing of WNV-WT and WNV-poly(A) to normal cells and tumor cells, further The killing specificity of WNV-poly(A) to tumor cells was determined by comparing the growth curves of WNV-WT and WNV-poly(A) on normal cells and tumor cells ( image 3 B). in:

[0144] Control group (MOCK): tumor cell group cultured in medium without virus;

[0145] Experimental group: the tumor cell group culture...

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Abstract

The present invention relates to an oncolytic virus and use thereof, the oncolytic virus comprising an attenuated flavivirus, the attenuated flavivirus comprising a polyadenylic acid (poly (A)) sequence, wherein the poly (A) sequence is used for replacing a part of nucleotide sequence of a 3' untranslated region (3' UTR) of the yellow disease virus, so that the 3' untranslated region (3' UTR) of the attenuated yellow disease virus obtained after the part of nucleotide sequence of the yellow disease virus is replaced at least retains a 3'-end stem-loop region (3' SL). In-vitro cell experiments prove that the oncolytic virus disclosed by the invention can infect and kill various tumor cells, but has small influence on normal cells; and a xenotransplantation tumor model proves that the oncolytic virus disclosed by the invention can inhibit the growth of various tumor cells in vivo, and a safe and efficient anti-cancer therapeutic agent with small toxic and side effects can be provided for clinical tumor treatment.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a broad-spectrum oncolytic application of attenuated West Nile virus. Background technique [0002] Tumor is the biggest public health problem facing human beings and the second leading cause of death. Malignant tumors have become one of the major public health problems that seriously threaten the health of people in China and the world. At present, the treatment for tumor mainly includes two types, one is traditional treatment including chemotherapy / radiotherapy / surgery, and the other is immunotherapy based on cytokines / immune cells and antibodies. However, no matter it is traditional treatment or immunotherapy, there are treatment defects. The biggest shortcoming of traditional treatment is drug resistance, while the biggest shortcoming of immunotherapy is the low response rate. Therefore, new treatment methods, such as tumor immunotherapy / oncolytic virus, ha...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/24C12N15/26C12N15/19C12N15/20C12N15/21C12N15/27C12N15/12C12N15/40C12N15/54C12N15/55C12N5/10A61K35/768A61K39/00A61K48/00A61P35/00C12R1/93
CPCC12N7/00C07K14/55C07K14/5412C07K14/5434C07K14/5443C07K14/54C07K14/521C07K14/555C07K14/56C07K14/535C07K14/70532C07K14/495C07K14/50C07K14/475C07K14/4703C12N9/1211C12N9/78C12N9/1077A61K35/768A61K39/0011A61K48/005A61P35/00C12Y207/01021C12Y305/04001C12Y204/02009C12N2770/24021C12N2770/24032A61K2039/5254A61K2039/5256A61K2039/876A61K2039/844A61K2039/86A61K2039/812A61K2039/892Y02A50/30
Inventor 张波袁绍鹏刘静占顺利高磊
Owner BEIJING SHUNLEI BIOTECHNOLOGY CO LTD
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