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Tumour bi-target adenovirus AdCN103 and its construction method and application

An oncolytic adenovirus and dual-targeting technology, which is applied in the fields of biotechnology and gene therapy, can solve problems such as mutation and potential safety hazards of oncolytic adenovirus, and achieve the effect of inhibiting tumor growth, an excellent technical platform, and easy to master

Inactive Publication Date: 2008-02-20
钱程 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these two methods also have their own defects: hTERT is also highly expressed in stem cells; certain virus infections can cause mutations in the Rb pathway of normal cells (such as hepatitis B virus HBV infection can cause mutations in the Rb pathway of normal liver cells)
Therefore, there are certain hidden dangers in the safety of single-targeted oncolytic adenoviruses. Based on this research, in order to ensure the safe use of oncolytic adenovirus vectors in clinical practice, we constructed a new generation of dual-targeted oncolytic adenoviruses. viral vector

Method used

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  • Tumour bi-target adenovirus AdCN103 and its construction method and application
  • Tumour bi-target adenovirus AdCN103 and its construction method and application
  • Tumour bi-target adenovirus AdCN103 and its construction method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1 1. Construction of a replicative oncolytic adenovirus carrying a novel hTERT promoter hTE1

[0040] A related plasmid construction

[0041] First design the following primers:

[0042] 255-P1: 5'-CTGCGCTGTCGGGGCCAGGC-3'

[0043] 255-P2: 5'-ATCGGCCAGGGCTTCCCACG-3'

[0044] 255-4DEB-P2: 5'-CCCACGTGCGCCCACGTGCGCCCACGTGCGATCGGCCAGGGCTTCCCACG-3'

[0045] 408-P1: 5'-CCACATCATGGCCCCTCCCT-3'

[0046] 408-P2: 5'-GCTGCCTGAAACTCGCGCCG-3'

[0047] 1125-P1: 5'-CTGTCCTGCGGTTGTGCCGG-3'

[0048] Using the PCR method, using the DNA of human embryonic kidney cells HEK293 as a template, primers 255-P1, 255-P2, to obtain the PCR product hTERT1, which is the hTERT promoter core -255 ~ +40nt sequence, and primers 408-P1, 408-P2 to obtain The PCR product hTERT3 is hTERT promoter core -408~+40nt sequence, primers 1125-P1, 408-P2, the PCR product hTERT3 is hTERT promoter core -1125~+40nt sequence

[0049] Using the PCR method, primers 255-P1, 255-4DEB-P2, using hTERT1 as a tem...

Embodiment 2

[0059] Example 2 , Evaluation of the activity of the novel hTERT promoter hTE1 in cells

[0060] The reporter gene plasmid pCMVβ-ga1 was purchased from Clontech Company, expressing β-galactosidase under the control of the CMV promoter; the plasmid pGL3-Basic (without promoter and enhancer), pGL3-Control ( containing SV40 promoter / enhancer) was purchased from Promega.

[0061] The four hTERT promoters were digested with KpnI+BglII and cloned into pGL3-Basic plasmid, named pGL3-BT-255, pGL3-BT-2554DEB, pGL3-BT-418, pGL3-SE. In this plasmid, the hTERT promoter drives the expression of the firefly luciferase gene. cells by 1.0 x 10 5 After inoculation in 6-well culture plates, 24 hours later, the cells were transfected with 1 μg of the plasmid containing the firefly luciferase reporter gene and 0.1 μg of the internal standard β-galactosidase reporter gene pCMV-βGal with LipofectAMINE (GIBCO BRL). After 48 hours, the cells were collected, washed twice with PBS, and lysed in Re...

Embodiment 3

[0063] Example 3 , Evaluation of the activity of the novel hTE1 promoter in nude mice

[0064] Every four to six Balb / c nude mice is a group, a total of six groups. Dissolve 20ug of plasmid DNA in 1.6ml of normal saline and inject into nude mice intravenously. After 24 hours, the nude mice were sacrificed, the liver was collected, and the liver was homogenized with 1.5 ml of PLB (Promega) solution containing Ultra-Turrax T25. The supernatant solution after centrifugation at 14,000 rpm at 4° C. for 10 minutes or after filtration was collected, and the fluorescence activity of each group of samples was measured.

[0065] As shown in Figure 3, only pGL3-BT-2554DEB could not detect fluorescence expression in nude mouse liver at all, indicating that its hTE1 promoter tumor targeting is very significant, and it has basically no toxic side effects on normal liver tissue.

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Abstract

The invention pertains to biological technique and gene treatment field, and discloses a dual tumor targeting replicative oncolytic adenouiruses, which is dually targeting, and controls the expression of virus early protein EIA and replication of virus by the synergism of specific tumor promoter and a CR2 region of a target deleted Rb protein. The invention also discloses a novel a specific tumor promoter hTERT-4DEB and a construction method thereof, the promoter adds three E-boxes behind an hTERT promoter -255-+40nt nuclear region, thus specifically enhancing the tumor target of the promoter. The invention takes AdCN103 as an example, and the oncolytic adenouiruses controls the expression of the CR2 region deleted E1A protein by modified hTERT-4DEB promoter, so the replicative oncolytic adenouiruses has dual targets. The invention also discloses a construction method of the use in preparing medicines for tumor treatment of the dual tumor targeting replicative oncolytic adenouiruses AdCN103; the dual tumor targeting replicative oncolytic adenouiruses AdCN103 can be used for the treatment of a plurality of tumors, which can be a new effective virus-gene anti-cancer medicine for the treatment of tumor.

Description

technical field [0001] The invention belongs to the fields of biotechnology and gene therapy, and in particular relates to a tumor dual-targeting virus vector AdCN103 specifically proliferating in tumor cells and its construction method and application. Background technique [0002] For most types of cancer, traditional therapies such as surgery, radiotherapy, chemotherapy, etc. are still used at present, but in the face of many advanced malignant tumors, traditional therapies are helpless, mainly manifested in the low cure rate, high recurrence rate and mortality , The prognosis is poor. Gene therapy is a biological high-tech solution to treat diseases by modifying genes. People are very enthusiastic about gene therapy for tumors, and 66% of gene therapy clinical trials are used for tumor treatment. So far, tumor gene therapy research has made important progress. For malignant tumors, gene therapy can use the molecular level differences between tumor cells and normal cells...

Claims

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Application Information

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IPC IPC(8): C12N15/861
Inventor 钱程蔡荣张唯
Owner 钱程
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