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Oncolytic adenovirus with target for liver cancer and its preparation method and use

A technology of oncolytic adenovirus and adenovirus, which is applied in the field of recombinant adenovirus, can solve the problems that have not been seen in liver cancer gene therapy of liver cancer, the impact of liver reserve ability, etc., and achieve high safety , high efficiency effect

Inactive Publication Date: 2005-04-06
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in this retroviral vector-mediated suicide gene therapy for liver cancer, due to the "bystander effect", liver stem cells will inevitably be affected, and liver reserve capacity will be affected
[0009] After searching, there are currently only retroviral vectors constructed abroad containing AFP alpha-fetoprotein and HRE hypoxia response elements, and adenoviral vectors without liver cancer-targeting deletion of the E1 region. A report on the gene therapy of liver cancer with replicating oncolytic adenovirus

Method used

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  • Oncolytic adenovirus with target for liver cancer and its preparation method and use
  • Oncolytic adenovirus with target for liver cancer and its preparation method and use
  • Oncolytic adenovirus with target for liver cancer and its preparation method and use

Examples

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Effect test

Embodiment 1

[0078] Example 1 Cloning of HRE and AF0.3 and Construction of Hybrid Promoter HREAF

[0079] 1. Find out the sequences of human vascular endothelial growth factor VEGF upstream hypoxia response element HRE and human α-fetoprotein core promoter AF0.3 from Genbank, and analyze the internal restriction sites

[0080] (1) For the HRE sequence containing the hypoxia response element, see GenBank data accession no. M63971. The approximately 380 bp DNA fragment between SacI and PstI (1188-1572) in the sequence is the hypoxia response element HRE.

[0081] (2) See GenBank data accession no. L34019 for the sequence containing α-fetoprotein core promoter AF0.3, and the 259 bp DNA fragment between 723 and 981 in the sequence is the AFP core promoter AF0.3. The PCR primers for the hypoxia response element HR and AFP core promoter AF0.3 were designed, and the hypoxia response element HRE primers: see sequence 1 in the sequence listing for the upstream primer, and refer to sequence 2 in...

Embodiment 2

[0100] Example 2 Adenovirus shuttle plasmid pShuttle-HREAF-E1 and

[0101] Construction of pShuttle-HREAF-E1Δ55

[0102] 1. Design the primers required for the construction of the shuttle plasmid

[0103] According to the hypoxia response element HRE (GenBank data accession no.M63971), AFP core promoter AF0.3 (GenBank data accession no.L34019), adenovirus E1a and E1b (GenBank data accession no.X02996) and shuttle reported in the literature Sequences at both ends of the multiple cloning site (MCS) of the plasmid pShuttle (Stratagene website www.stratagene.com), design primers respectively, and add different restriction internals to the 5' end or 3' end of the plasmid depending on the needs of vector construction Dicer sites are used for PCR amplification of each construction element, and primers are synthesized by Sebassen.

[0104] HREAF primers: the upstream primer is shown in sequence 6 in the sequence listing, with a KpnI site added, and the downstrea...

Embodiment 3

[0119] Example 3 Preparation of recombinant oncolytic adenovirus AdhafE1Δ55

[0120] 1. AdEasy XL adenoviral vector system was used to produce recombinant adenovirus ( Figure 5 )

[0121] After the shuttle plasmid was constructed, it was linearized with PmeI, and transformed into BJ5183 (product of Stratagene) competent bacteria together with the backbone plasmid pAdEasy-1. The two plasmids were homologously recombined in the bacteria, and the recombinant adenovirus plasmid was selected by kanamycin resistance, and then identified by PacI digestion in vitro. The recombinant adenovirus plasmid should be digested with PacI to produce a large fragment of about 30kb, and a small fragment of 3.0kb (recombination occurs in the left arm) or 4.5kb (recombination occurs in the pBR322 origin of replication). Recombinant adenovirus plasmids without PacI digestion, on the 0.8% agarose gel electrophoresis band, appear as a fuzzy band at the top near the sample well, and often the...

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Abstract

The invention discloses a kind of liver cancer target tropism to dissolve the lump adenovirus, its preparation method and use. The invention adenovirus is starts sub- AF0.3 using oxygen deficit reply part HRE and the AFP core to construct mixed and the start, constructs the retention to have the E1 duplication start area gene the reorganization adenovirus shuttle material particle, again constructs the reorganization adenovirus material particle after the homologous reorganization, the extension dyes the cell, the packing leaves the goal reorganization virus pellet. The invention adenovirus has the liver cancer target tropism, can oneself duplicate in the liver cancer cell, achieves the decomposition cancer cell the goal. Has highly effective, not sends the sudden change function, the safe highly effective merit, may use in to prepare the cancer cell the treatment medicine.

Description

technical field [0001] The invention relates to a recombinant adenovirus, in particular to a liver cancer-targeting oncolytic adenovirus, and also to a preparation method and application of the virus. Background technique [0002] The morbidity and mortality of liver cancer have been high in my country, and it is the main tumor that endangers the health of the Chinese population, but there is no specific treatment. If an effective liver cancer biotherapeutic agent can be developed, it is not only of great significance to explore the treatment of liver cancer, but also has obvious social and economic benefits. [0003] The occurrence and development of tumors is often the result of synergistic effects of multiple genes, and gene therapy methods that only restore the function of one or several tumor suppressor genes are generally difficult to be effective. " published an editorial in the journal, and firstly proposed a new strategy of using tumor selective replication Adenovi...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61P35/00C12N7/01C12N15/11C12N15/63C12N15/861C12Q1/70
Inventor 陆应鳞陈泽建杜芝燕徐丁尧蔺会云徐元基王妍
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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