HIL7/hCCL19 double-gene recombinant oncolytic virus as well as preparation method and application thereof

An oncolytic virus and genome technology, applied in the field of genetic engineering, can solve the problems of reduced tumor sensitivity, lower virus killing and replication ability than wild-type virus, etc.

Active Publication Date: 2022-07-08
SUNSHINE LAKE PHARM CO LTD
View PDF2 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Carrying multiple exogenous genes can effectively make up for the limited anti-tumor effect of oncolytic viruses. The sensitivity of viruses to tumors is often significantly reduced. For example, studies by Mckie E A and others have shown that it can be reduced by 100 times (Mckie E A, Maclean A R, Lewis A D, et al. Selective in vitro replication of herpes simplex virus type 1 (HSV-1) ICP34. 5 null mutants in primary human CNS tumours--evaluation of apotentially effective clinical therapy.[J].British Journal of Cancer,1996,74(5):745-752.)

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • HIL7/hCCL19 double-gene recombinant oncolytic virus as well as preparation method and application thereof
  • HIL7/hCCL19 double-gene recombinant oncolytic virus as well as preparation method and application thereof
  • HIL7/hCCL19 double-gene recombinant oncolytic virus as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1 Construction of recombinant virus

[0093] Insert the gene sequences encoding hIL7 and hCCL19 between the homology arm sequences on the left and right sides of the ICP34.5 gene to obtain the pMD18T-HOM-hIL7-hCCL19 donor plasmid, as shown in the appendix. figure 1 , the ICP34.5 gene of the HSV-1 / ICP47-knockout virus strain was replaced by the therapeutic genes hIL7 (human IL7) and hCCL19 (human CCL19) by the Crisper-Cas9 system, the recombinant virus was constructed, and the successfully constructed virus was screened. The first nucleotide of the start codon of the ICP34.5 gene is used as the first nucleotide for sequence coding, and the gene insertion position is between the 134th nucleotide of the start codon and the 160th nucleotide downstream of the stop codon. between nt522-nt1271 and nt124324-nt125073 in the wild-type HSV-1 genome (GeneBank: JQ780693.1).

[0094] Using the Crisper-Cas9 gene editing system to cut the Target DNA (targeted DNA) of the oncol...

Embodiment 2

[0107] Example 2 PCR verification

[0108] According to the virus titer, the virus (recombinant virus obtained in Example 1) was infected with VERO cells covered with six-well plates, the size of virus plaques was observed, and DMEM medium containing 0.01% neutral red was added for staining. Monoclonal virus plaques were amplified and the genome was extracted for PCR verification.

[0109] (1) Experimental materials:

[0110] Virus monoclonal Δ47-hIL7-hCCL19-1 and Δ47-hIL7-hCCL19-2 after multiple rounds of screening.

[0111] (2) PCR verification: the primer is HOM1-F / 34.5-flank-seq, the PCR product size is 3975bp,

[0112] HOM1-F: 5'-CGCACAGTCCCAGGTAACCTC-3', as shown in SEQ ID NO:8;

[0113] 34. 5-flank-seq: 5'-GGCGTGTCTCTGTGTATGAGTCAGGGGGTCC-3' as shown in SEQ ID NO:9.

[0114] PCR system

[0115]

[0116]

[0117] PCR program: 98°C, 3min; (98°C, 10s; 55°C, 15s; 68°C, 3min) x 35 cycles; 72°C, 5min; 12°C, forever.

[0118] (3) Sequencing results and ana...

Embodiment 3

[0119] Example 3 Protein ELISA identification

[0120] (1) Experimental materials:

[0121] The experimental group Δ47-hIL7-hCCL19 refers to the Δ47-hIL7-hCCL19-1 recombinant oncolytic virus, and the negative control is the Δ47 oncolytic virus.

[0122] (2) ELISA verification: Vero (African green monkey kidney cells) was used as the host cell, and the conditions were as follows: Δ47-hIL7-hCCL19, Δ47 infection MOI (ratio of virus to cell number) was 0.01, after infection with Vero cells, 24 , 48, 72 hours to collect the cultured virus culture supernatant.

[0123] (3) hCCL19 expression results:

[0124] The expression results of hCCL19 in Vero cells are shown in Table 1.

[0125] Table 1:

[0126]

[0127] The expression results of hCCL19 in Vero cells are as follows Figure 4 As shown, the expression of hCCL19 protein at three time points (24h, 48h, 72h) is shown. The figure shows that with the increase of infection time, the expression of hCCL19 first increases and th...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to the field of gene engineering, in particular to an hIL7 / hCCL19 double-gene recombinant oncolytic virus as well as a preparation method and application thereof. According to the recombinant oncolytic virus provided by the invention, two genes IL7 and CCL19 with a relatively strong synergistic effect are integrated in a genome of the recombinant oncolytic virus, the effect of the oncolytic virus is enhanced, and compared with an oncolytic virus of which the sensitivity is generally reduced after modification, the double-gene carrying type virus constructed by the invention keeps the sensitivity to tumor cells, and can be used for preparing the oncolytic virus. Meanwhile, the virus replication capability is not reduced.

Description

technical field [0001] The present invention relates to the field of genetic engineering, and in particular, the present invention relates to a hIL7 / hCCL19 double-gene recombinant oncolytic virus and a preparation method and use thereof. Background technique [0002] The biggest problem facing the current oncolytic virus is the curative effect. The anti-tumor effect of oncolytic virus is remarkable, but the effect is not ideal, and the T-vec product approved by the FDA, the overall response rate of the oncolytic treatment group is only 26% %, how to transform the oncolytic virus or use it in combination to completely eliminate the tumor is the main work of most researchers. Due to the molecular nature of CCL19, repeated high-concentration administration is required, and it cannot be well applied due to large side effects. The viral vector is used to carry the CCL19 factor for continuous local expression to avoid rapid clearance of CCL19. Carrying multiple exogenous genes ca...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N7/01C12N15/85C12N15/90C12N15/24C12N15/19A61K38/20A61K38/19A61K48/00A61K35/763A61P35/00C12R1/93
CPCC12N7/00C12N15/85C12N15/907C07K14/5418C07K14/523A61K38/2046A61K38/195A61K48/0008A61K48/005A61K35/763A61P35/00C12N2710/16621C12N2800/107C12N2710/16652C12N2710/16632C12N15/86C12N2710/16643C07K14/521Y02A50/30A61K2300/00
Inventor 张旭辉罗显麟陈小锋李文佳
Owner SUNSHINE LAKE PHARM CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap