Non-reproductive recombinant herpes virus and use thereof

A non-replicating, herpes simplex virus technology, applied in the field of non-replicating recombinant herpes virus, can solve the problems such as transfection and expression of foreign genes need to be improved

Active Publication Date: 2021-06-29
BEIJING WELLGENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Most of the non-replicating herpes viruses currently in use are derived from laboratory virus strains that have been passaged in tissue culture cells for many years. ability to be improved

Method used

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  • Non-reproductive recombinant herpes virus and use thereof
  • Non-reproductive recombinant herpes virus and use thereof
  • Non-reproductive recombinant herpes virus and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] The acquisition of embodiment 1 HL-1 strain virus

[0088] a) Virus collection

[0089] 63 healthy volunteers with recurrent herpes labialis were recruited, and the herpes fluid was aseptically collected.

[0090] b) Virus screening

[0091] The purpose of this section is to test and screen primary clinical isolates of HSV-1 and select the virus strain with the strongest oncolytic ability.

[0092] Experiments were performed simultaneously using 5 to 8 virus strains each time. Using 17 strains of HSV-1 virus as a control, the virus strains were infected with the same MOI in cells in a six-well plate, and the experiments were performed in parallel. After 48 hours of infection, the CPE phenomenon caused by the virus was observed and the number of plaques after crystal violet staining was evaluated. . CPE should be caused by typical HSV-1 infection. The cells become round and gradually fall off from the center of infection to form plaques. Strains with a pronounced CP...

Embodiment 2

[0105] The contrast of embodiment 2 wild HL-1 strains and 17 strains

[0106] This example provides the comparative experiments and results of replication of HL-1 and 17 strains on different cells and their ability to transfect tumor cells. Experimental cells include: Hep G2 human liver cancer cells, A-375 human skin cancer cells, A549 human lung cancer cells, MCF7 human breast cancer cells, HeLa human cervical cancer cells, U251 human glioma cells, and Vero monkey kidney cells.

[0107] The experimental method is as follows:

[0108] 1. HL-1 and HSV(17) were transfected into different cells and observed under microscope

[0109] The above-mentioned cells in the logarithmic growth phase were taken, digested and subcultured into a six-well plate for culture. After 24 hours, the old medium was discarded, and 2ml of medium was added to each well. The transfection was carried out according to the number of viruses at different MOIs. After the virus was added, it was placed in a...

Embodiment 3

[0132] The contrast of embodiment 3 non-replication HSV (HL-1) virus and HSV (17) virus infecting nerve cell ability

[0133] The purpose of this example is to compare the ability of non-replicative HL-1 viruses knocked out of different immediate genes and 17 strains to infect nerve cells, so as to compare the non-replicative HL-1 herpes virus of the present invention as a gene carrier with the standard in the prior art The effect of 17 strains of laboratory virus.

[0134] Construct non-replicative viruses according to methods 1 and 2, respectively, as follows:

[0135] 1. Knock out the ICP34.5 and ICP4 genes of HL-1 strain and 17 strains of HSV virus according to the method of application publication number CN105219739A, and insert an exogenous reporter gene in the LAT region of the virus. From the 5' end to the 3' end, the gene includes: CMV promoter, gene encoding LacZ and BGH PolyA.

[0136] 2. Knock out the ICP34.5 and ICP4 genes of the HL-1 strain and 17 strains of HS...

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Abstract

The application relates to a non-reproductive herpes simplex virus. One or more immediate early genes are knocked out or destroyed, and thus, reproduction capability is lost; and the herpes simplex virus is derived from a herpes simplex virus with an accession number of CCTCC V201810 or descendants thereof. The invention further relates to a composition containing the virus and a viral culture of the composition.

Description

technical field [0001] The present invention relates to non-replicating recombinant herpes virus and its use. The expression of one or more immediate early genes of the non-replicating recombinant herpesvirus is prevented or reduced by gene mutation, thereby losing the ability to replicate. The non-replicative recombinant herpes virus can be used as a non-replicative viral vector to introduce foreign genes into subjects, such as subjects suffering from nervous system diseases and blood system diseases, so as to treat these related diseases. Background technique [0002] Herpes Simplex Virus (HSV) belongs to the Herpesviridae family and is an enveloped double-stranded DNA virus, including two subtypes, type I (HSV-1) and type II (HSV-2). [0003] Herpes simplex virus consists of four parts: core, capsid, interlayer protein and envelope. The viral core consists of thick double-stranded DNA wound up into a filamentous scroll. The capsid surrounded by DNA has an icosahedral s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/04
CPCC12N7/00C07K14/005C12N2710/16621C12N2710/16622C12N2710/16662C12N2710/16632C12N2710/16643A61K35/763
Inventor 赵婧姝李小鹏田超周华王田田刘家家孙春阳闻可心
Owner BEIJING WELLGENE CO LTD
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