Use and constructing plan of anticancer recombined gland virus with tumour CHK1 as target of medicine

A technology of recombinant adenovirus and construct, which is applied in the field of medical genetic engineering, can solve the problems of limited therapeutic effect, lack of tumor specificity, and no therapeutic target gene, and achieve the effect of avoiding attack and increasing copy number

Inactive Publication Date: 2004-12-08
深圳市天达康基因工程有限公司
View PDF5 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the second-generation ADV5 gene therapy vectors have some common disadvantages: (1) Although the vectors designed based on the biological basis of adenoviruses have the therapeutic advantage of replicability in tumor conditions, due to various design deficiencies, the obtained adenoviruses are The efficacy of replication and lysis of tumor cells in tumor cells is far weaker than that of wild-type ADV5. For example, the anticancer effective rate of ONYX-015 alone is only 0-14%
(2) The application of the second-generation ADV5 gene therapy vector is still based on local tumor injection, which is greatly limited in application. In most cases, tumor is a systemic disease and needs to be administered intravenously Systematic treatment is carried out to control primary and metastatic lesions. Therefore, the selectivity of the second-generation ADV5 gene therapy vector and the killing efficiency of tumor cells must be further enhanced to meet the needs of improving drug delivery routes
(3) None of the second-generation ADV5 gene therapy vectors carry effective thera

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
  • Use and constructing plan of anticancer recombined gland virus with tumour CHK1 as target of medicine
  • Use and constructing plan of anticancer recombined gland virus with tumour CHK1 as target of medicine
  • Use and constructing plan of anticancer recombined gland virus with tumour CHK1 as target of medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Construction of pXC1 series mutants (Δ923-946pXC1)

[0031] 1. pXC1 was purchased from Microbix Biosystem Inc. (Toronato, Ontario, Canada, catalog number: PD-01-03). The plasmid contains the 21-5790nt sequence of human adenovirus type 5 (ADV5). The plasmid structure is shown in Figure 1 194-358nt of the plasmid is the ADV5 adenovirus packaging signal, the sequence 560-1112nt, 1229-1545nt is the coding sequence of functional protein E1A, 9883-9888nt contains the BamHI restriction site, and 1338-1343nt contains the XbaI restriction site.

[0032] 2. Deletion of 923-946nt by 3 times PCR.

[0033] (1) Acquisition of Fragment 1: Primer 1 = 5'-CG GGA TCC GGG CCC CCA TTT CC-3' (equivalent to 9883-9902nt, the underlined part is the BamHI restriction site); primer 2 = 5'- GTC ACT GGG TGG ATC GAT CAC CTC CGG TAC-3' (equivalent to 922-905nt, the underlined part is complementary to primer 3);

[0034] Use pXC1 as a template for PCR reaction, the total volume of t...

Embodiment 2

[0042] Example 2: Construction of Δ923-946ADV5 recombinant adenovirus

[0043] 1. pBHGE3 was purchased from Microbix Biosystem Inc. (Toronato, Ontario, Canada, catalog number: PD-01-12). This plasmid contains all genome sequences except the ADV5 packaging signal (194-358nt), and between 194-358nt An artificial sequence is inserted between them, the full length is 37436bp, and its structure is shown in Figure 2.

[0044] When pBHGE3 is obtained from Microbix Biosystem Inc., the total amount is 10 μg. First, electroporate into competent bacteria and pick positive clones. Since the plasmid fragment is very long, CsCl is needed 2 -Eb The plasmid was purified by ultracentrifugation according to the method in the book "Molecular Cloning".

[0045] 2. Homologous recombination method to obtain Δ923-946ADV5 recombinant adenovirus construct, the method is as follows:

[0046] (1), plant 7.5×10 in a 15cm petri dish 5 293 cells (ATCC, U.S.A, catalog number: CRL-1573), the culture mediu...

Embodiment 3

[0062] Example 3: Construction of the subcloning vector pCDNA3.1-ΔE3 in the ADV5 E3 region

[0063] 1. The full name of the ADV5 E3 region is the early region 3 of ADV5. Driven by the endogenous promoter, this region encodes 7 proteins. The sequence, structure and function are as follows (see Figure 3): 12.5k, 27858-28179nt, function Unidentified; 6.7k, 27547-28736nt, function unknown; gp19k, 28735-29215nt, binds MHC class I antigens, inhibits their presentation to the cell surface, resulting in evasion of CTL clearance; ADP, 29419-29770nt, lyses cells, releases adenovirus ; RIDα, 29784-30057nt, forms a complex with RIDβ, prevents the lysis of TNF, and clears the FAS antigen; RIDβ, 30062-30458nt; 14.7k, 30453-30837, inhibits the lysis of TNF.

[0064] The purpose of this experiment is to delete the 28530-29355nt region of the E3 region and insert an exogenous therapeutic gene into the E36.7k / gp 19k region of the recombinant adenovirus.

[0065] 2. Delete 28532-29360nt of ADV5...

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

This invention relates to establishment of artificially reformed human adenovirus 5 and use in treatment of cancers by reorganized configuration of adenovirus. It is obtained by extension of fixed-point deficiency, homologous reorganization, transfection, and monoclone purification of adenovirus. Its novelty includes deletion of 24 alkali groups in encoding sequence of ADV 5 gene set E1A conservative sequence 2, deletion of 28532 - 29360nt of ADV 5 E3 with reversed inserting some of CHK1 cDNA segments. The reorganized configuration of adenovirus can selectively regenerate in tumors, with specific expression of reversed CHK1 cDNA segments to kill tumor cells but normal cells. The configuration is unique effectiveness in diagnosis and treatment of tumors. It provides an ideal therapy of gene target specificity.

Description

1. Technical field [0001] The present invention relates to a human adenovirus type 5 (Human adenovirus type 5, ADV5) recombinant construction scheme, which is characterized in that: directional deletion of the 923-946nt sequence of the ADV5 genome (CTTACC TGC CAC GAG GCT GGC TTT, the sequence codes E1A protein 122 to 129 amino acids), on this basis, further delete the ADV5 E3 region 28532-29360nt, introduce a ClaI restriction site in the deletion region; reversely insert a 620bp exogenous CHK1 cDNA fragment (equivalent to 853-250nt of CHK1 mRNA), so as to obtain the recombinant adenovirus construct Δ923-946ADV5 / ASCHK1 which has a therapeutic effect on tumors. The invention belongs to the technical field of medical genetic engineering. 2. Background technology [0002] Malignant tumors are becoming the primary disease that endangers human health. According to the latest domestic epidemiological survey data, there are more than 3 million cancer patients in my country, about ...

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): A61K48/00A61P35/00C12N7/01C12N15/861
Inventor 周剑峰马丁卢运萍王世宣陈刚高庆蕾
Owner 深圳市天达康基因工程有限公司
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