New genetically-modified vaccinia virus

A technology of vaccinia virus and composition, which is applied in the field of new genetically recombined vaccinia virus, and can solve the problems such as the revelation that there are no multiple therapeutic gene combinations

Active Publication Date: 2019-03-15
ASTELLAS PHARMA INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • New genetically-modified vaccinia virus
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  • New genetically-modified vaccinia virus

Examples

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Embodiment 1

[0164] (Example 1: Construction of Transfer Vector Plasmid DNA)

[0165] The transfer vector plasmid DNA used for producing recombinant vaccinia virus by the homologous recombination method was prepared as follows.

[0166] (1) Construction of pTN-VGF-P-DsRed transfer vector plasmid DNA

[0167] The pUC19-VGF vector was prepared based on International Publication No. 2015 / 076422. More specifically, for the preparation of pUC19-VGF vector, genomic DNA of LC16mO strain (accession number AY678277.1) was used as a template, and pUC19 vector (product code: 54357) of Invitrogen Corporation was used. The prepared pUC19-VGF vector was cleaved with restriction enzyme AccI, and the ends were blunted. A DNA fragment (SEQ ID NO: 22) containing the p7.5k promoter and the DsRed fragment was inserted into the cleavage site, thereby constructing a transfer vector plasmid DNA. The constructed plasmid DNA was called pTN-VGF-P-DsRed.

[0168] (2) Construction of pTN-VGF-SP-IL12 and pTN-VGF...

Embodiment 2

[0174] (Example 2: Construction of genetically recombinant vaccinia virus)

[0175] A recombinant vaccinia virus (called LC16mOVGF-SP-LucGFP / O1L-p7.5-DsRed) deficient in the functions of VGF and O1L was produced from the vaccinia virus LC16mO strain. For this virus, use the next generation sequencer PacBio RSII (Pacific Bioscience Company) to carry out sequencing, use Sprai [BMC GENOMICS.2014 AUG 21; 15:699.] software to carry out the reconstruction of viral genome from the obtained sequence information to determine the base As a result, it has the base sequence represented by SEQ ID NO: 21. In addition, loop sequences are added to both ends of the base sequence, and the loop sequences at both ends are the base sequences represented by SEQ ID NO: 19 or 20.

[0176] (1) Recycling has figure 2 Recombinant vaccinia viruses with viral genomes indicated. Hereinafter, the recovery step will be specifically described. Make LC16mO VGF-SP-LucGFP / O1L-p7.5-DsRed infect CV1 cells (...

Embodiment 3

[0182] (Example 3: Tumor lysis of genetically recombinant vaccinia virus)

[0183] The hIL12- and hIL7-carrying vaccinia viruses prepared in Example 2 were evaluated for their lytic ability (cell death ability) in various human cancer cells. Furthermore, the lytic ability in various human cancer cells was similarly evaluated for the combination mixture of two viruses, hIL12-carrying vaccinia virus and hIL7-carrying vaccinia virus prepared in Example 2.

[0184] Specifically, first, in a 96-well plate (AGC Techno Class company), add the following medium (containing 10% fetal bovine serum (GE Healthcare company) and 1% penicillin streptomycin (Life Technology company) culture medium) to reach 1×10 4 100 μL of each cell suspended in a cell / mL format. After culturing overnight, a mixture of 1) hIL12 and hIL7-carrying vaccinia virus and 2) hIL12-carrying vaccinia virus and hIL7-carrying vaccinia virus at a concentration of 1:1 was mixed using Opti-MEM (Life Technology Co., Ltd....

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Abstract

The invention provides a genetically-modified vaccinia virus that is effective for the prevention or treatment of cancer. Specifically, the invention provides: a vaccinia virus containing two polynucleotides which are a polynucleotide that codes for IL-7 and a polynucleotide that codes for IL-12; a combination kit including two vaccinia viruses which are a vaccinia virus containing a polynucleotide that codes for IL-7 and a vaccinia virus containing a polynucleotide that codes for IL-12; and a combination use of the two vaccinia viruses.

Description

technical field [0001] The invention relates to a novel gene recombinant vaccinia virus. Background technique [0002] In recent years, various techniques for using viruses for cancer therapy have been developed, and one of the viruses used for cancer therapy is vaccinia virus. Vaccinia virus is used as a carrier for delivering therapeutic genes to cancer cells, as a tumor-lytic virus that proliferates in cancer cells and destroys cancer cells, or as a cancer vaccine that expresses cancer antigens or immunomodulatory molecules, for the purpose of cancer treatment Research was conducted (Expert Opinion on Biological Therapy, 2011, Vol. 11, p. 595-608). [0003] It has been reported that the N1L gene is inactivated by insertion of exogenous genes encoding interleukin-12 (IL-12) or interleukin-21 (IL-21), and by the LacZ reporter gene and the firefly luciferase gene. The vaccinia virus obtained by manipulating the thymidine kinase (TK) gene by inserting it into the thymidine ...

Claims

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Application Information

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IPC IPC(8): C12N15/09A61K35/768A61P35/00A61P35/02A61P37/04C12N7/01
CPCA61K35/768A61P37/04A61P35/00A61P35/02C12N15/86C12N2710/24143C12N2710/24132C12N7/00C12N2710/24162C12N2710/24121A61K38/2046A61K38/208A61K48/005C07K14/5418C07K14/5434C12N2710/24171C12N15/09
Inventor 中尾慎典川濑龙也中村贵史
Owner ASTELLAS PHARMA INC
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