Safe coxsackie virus for treating KRAS mutant tumors, and pharmaceutical composition thereof

A technology of coxsackie virus and composition, which is applied in the field of medicine and can solve the problem that therapeutic agents cannot be safely administered

Active Publication Date: 2020-04-21
REVOIMMUNE THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007]However, some tumor drugs show some obvious side effects, especially some candidate therapeutic agents (such as...

Method used

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  • Safe coxsackie virus for treating KRAS mutant tumors, and pharmaceutical composition thereof
  • Safe coxsackie virus for treating KRAS mutant tumors, and pharmaceutical composition thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Coxsackie virus preparation

[0103] 1.1 Sequence synthesis and plasmid construction:

[0104] The DNA sequence corresponding to the myocardial miRNA targeting sequence shown in Table 1 was synthesized by artificial synthesis method:

[0105] Table 1 DNA sequence of myocardial miRNA targeting sequence

[0106]

[0107] Containing Sfi I site, artificial 3C pro / 3CD pro Cleavage site and pMKS1 plasmid encoding full-length cDNA of CVB3 (Wodrolf strain) (available from J. Lindsay Whitton (The Scripps Research Institute, LaJolla, California)). The SfiI site in this plasmid serves as an accepting cloning site for insertion of the appropriate oligonucleotide sequence.

[0108] Complementary sequences containing three miRNAs (respectively denoted as miR-A, miR-B and miR-C) (i.e. myocardial miRNA targeting sequence) were generated by PCR, which were continuously incorporated into the 3'UTR of pMKS1 to generate plasmid pMKS1 - A-B-C (CVB3-miRNA). The resulting plasmid wi...

Embodiment 2

[0130] Symptoms of the virus

[0131] 2.1 Virus growth characteristics

[0132] For the one-step growth curve of the virus, it is determined as follows:

[0133] a) after inoculating HeLa cells to form a monolayer, infect HeLa cells with the virus CVB3 obtained in Example 1;

[0134] b) Place virus-infected cells at 37°C 5% CO 2 In the incubator, at different time points, the cell supernatant was collected to detect the virus titer;

[0135] c) Draw a change curve of the virus gradient with the culture time, that is, a one-step growth curve.

[0136] CVB3 is a cytolytic virus. The earlier its progeny virus appears in the cell culture supernatant, the faster the titer rises. The higher the titer, the lower the survival rate of sensitive cells, and the corresponding coxsackie virus mutant strain is cytolytic. The higher the ability.

[0137] 2.2 Cytotoxicity test

[0138] Different kinds of tumor cells were inoculated in 12-well plates. When the cell density reached 90%, r...

Embodiment 3

[0150] Performance testing of Coxsackie virus

[0151] 3.1 Western blotting:

[0152] In order to further evaluate whether the Coxsackievirus of the present invention can inhibit the synthesis of viral proteins in normal cell lines without restricting the viral replication in PC cells, the viral protein 1 (VP1) and cleaved caspase-3 (cells) were determined by Western blot analysis. A marker of death) protein expression levels. CVB3-miRNA infected cells will be harvested and lysed. Briefly, equal amounts of proteins will be subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes. The resulting membranes will be incubated with primary antibodies to VP1, cleaved Caspase-3 and β-actin. Protein levels of VP1 and cleaved caspase-3 will be quantified by densitometric analysis using NIH ImageJ and then normalized to β-actin.

[0153] The results show that the Coxsackie virus of the present invention hardly ...

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Abstract

The invention provides safe coxsackie virus for treating KRAS mutant tumors, and a pharmaceutical composition thereof, specifically coxsackie virus capable of specifically infecting tumor cells, wherein the coxsackie virus specifically infects tumor cells, and the ratio R1 (F1/F0) of the infection rate F1 of the coxsackie virus to normal myocardial cells to the infection rate F0 of wild type coxsackie virus subtype 3 to normal myocardial cells is less than or equal to 1/500. The coxsackie virus disclosed by the invention infects tumor cells with high specificity, and hardly infects normal cells, so that the cardiotoxicity of the coxsackie virus is greatly reduced and even eliminated.

Description

technical field [0001] The invention relates to the field of medicine, in particular to a safe coxsackie virus for treating KRAS mutant tumors and a pharmaceutical composition thereof. Background technique [0002] With the deepening of life science and medical research, gene therapy has become an important development direction of cancer targeted therapy, and oncolytic virus-mediated gene therapy is an effective strategy for cancer targeted therapy. In nature, only a few viruses are spontaneous oncolytic viruses, while most viruses require genetic modification to achieve targeted infection of tumor cells. Studies have shown that adenovirus, herpes simplex virus, enterovirus and measles virus have been successfully transformed into conditional replication viruses with oncolytic properties through molecular biological means, that is, oncolytic viruses. Oncolytic viruses refer to a class of therapeutic viruses that selectively infect and kill tumor cells. In the initial stag...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K35/768A61P35/00C12R1/93
CPCC12N7/00A61K35/768A61P35/00C12N2770/32021
Inventor 吴可行
Owner REVOIMMUNE THERAPEUTICS INC
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