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Recombinant herpes simplex virus and method for preparing same

A herpes simplex virus and genome technology, applied in biochemical equipment and methods, viruses, virus/bacteriophage, etc., can solve the problems of virus replication limitation, difficult genetic manipulation, and low therapeutic efficacy

Pending Publication Date: 2021-03-19
SUNG KWANG MEDICAL FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most current developments against cancer HSV-1 are limited by their low therapeutic potency due to the restriction of viral replication due to the loss of genes required for viral replication
In addition, since HSV-1 is a large DNA virus with a size of 152 kb, there is a problem that genetic manipulation is not easy

Method used

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  • Recombinant herpes simplex virus and method for preparing same
  • Recombinant herpes simplex virus and method for preparing same
  • Recombinant herpes simplex virus and method for preparing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Embodiment 1: Make wherein delete the recombinant HSV-1 of ICP6 and IR region (ΔICP6ΔIR HSV-1)

[0074] figure 1 A is a schematic diagram of the genome of HSV-1. The HSV-1 genome is mainly divided into a long unique (Unique Long: UL) region and a short unique (Unique short: US) region. In the UL region, a single origin of replication and a and b consisting of terminal repeats (Terminal repeat: TR) and IR are located at 5', while a and b in the reverse direction are located at 3'. In the US region, two replication origins and the c sequence in the reverse direction are located at the 5', the c sequence in the forward direction is repeated at the back, and the a sequence is repeated once at the 3' end.

[0075] First, a ΔICP6 HSV-1 virus was produced in which a part of the ICP6 region was deleted from the genome sequence of the wild-type HSV-1 WT virus. Nucleotides 86,364 to 89,777 in the wild-type HSV-1 genome sequence are sequences encoding the ICP6 gene. Nucleotide...

Embodiment 2

[0078] Example 2: Production of ΔICP6-ΔIR-rpsL-neo into which foreign gene rpsL-neo is inserted

[0079] A virus was produced by inserting rpsL-neo gene 1319 bp as a foreign gene into the ΔICP6ΔIR HSV-1 of Example 1 in which both the ICP6 gene and the IR gene were deleted, and it was verified whether the foreign gene was inserted.

[0080] Specifically, as shown in Table 1 below, the UL56-rpsL-neo primer with a sequence complementary to UL56 and rpsL-neo and the UsIR-rpsL-neo primer with a sequence complementary to UsIR and rpsL-neo were made with 200 ng of rpsL- The neo gene was used as a template, and PCR was performed using 10 pmol of UL56-rpsL-neo primer, UsIR-rpsL-neo primer and 2X green master mix (thermo scientific) reaction enzyme. The PCR product was transformed into DH10b Escherichia coli together with the ΔICP6 total vector of Example 1 to induce homologous recombination (homologous recombination), thereby producing ΔICP6-ΔIR-rpsL-neo expressing rpsL-neo.

[0081] ...

Embodiment 3

[0092] Example 3: Making ΔICP6ΔIR virus

[0093] The ΔICP6ΔIR virus was prepared by removing the foreign gene rpsL-neo from the ΔICP6-ΔIR-rpsL-neo prepared in Example 2 above.

[0094] Specifically, after preparing the primers in Table 3 below, a double-stranded oligomer bound with a complementary sequence was prepared by performing a slow cooling reduction method (primer annealing). The produced oligomer was transformed into DH10b Escherichia coli together with the ΔICP6-ΔIR-rpsL-neo total vector of Example 2 to induce homologous recombination, thereby producing dICP6-dIR.

[0095] 【table 3】

[0096]

[0097] In order to confirm whether the ΔICP6ΔIR virus from which the foreign gene was removed was successfully produced, PCR was carried out using the above-mentioned PCR primers. Using 200ng as a template, after adding 2X green master mix (thermo scientific) reaction enzyme to 10pmol of UL55 S primer and ΔIR AS primer, perform PCR in a PCR machine (Bio-rad) according to t...

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Abstract

The present invention relates to a recombinant herpes simplex virus type 1 (HSV-1), a recombinant HSV-1 vector, and a method for preparing same. The recombinant HSV-1 according to one aspect enables easy genetic modification, as a relatively large foreign gene can be inserted or various foreign genes can be inserted simultaneously, as ICP6 and an IR region are deleted simultaneously. In addition,the present invention can be variously utilized in the cancer treatment field as an oncolytic virus that is safe, while having an excellent effect for killing cancer cells.

Description

technical field [0001] The invention relates to a recombinant herpes simplex virus type 1 (HSV-1), a recombinant HSV-1 vector and a manufacturing method thereof. Background technique [0002] Herpes Simplex Virus (HSV) is a virus belonging to the family Herpesviridae, which is divided into HSV-1 and HSV-2, and known as human herpesvirus 1 and 2, respectively. Nucleic acid is double-stranded linear DNA with a molecular weight of 96×10 6 dalton. [0003] Since T-VEC, which was approved by the U.S. Food and Drug Administration (FDA) in 2015, is clinically used for malignant melanoma, development of a herpesvirus-based anticancer virus is underway. However, most currently developed anticancer HSV-1s are limited by their low therapeutic potency due to the limitation of viral replication due to the loss of genes required for viral replication. In addition, since HSV-1 is a large DNA virus with a size of 152 kb, there is a problem that genetic manipulation is not easy. [0004]...

Claims

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Application Information

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IPC IPC(8): C12N7/00
CPCC12N7/00C12N2710/16011C12N2710/16032C12N2710/16051
Inventor 崔璟珠金周恒
Owner SUNG KWANG MEDICAL FOUND
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