Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Complementary type high capacity adenoviral vector and vaccine and gene therapy producers constructed by the same

A vector system, adenovirus technology, applied in gene therapy, genetic engineering, plant genetic improvement, etc., can solve the problems of low yield, deletion and recombination, lack of selective growth advantages, etc.

Inactive Publication Date: 2009-02-11
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
View PDF0 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this system is that the growth of the high-capacity vector is completely dependent on the helper virus, which lacks the advantage of selective growth, so the yield is very low
The third-generation adenoviral vector can only be produced in cell lines with the help of helper virus, and the removal of helper virus in large-scale production and purification greatly increases the cost of production and purification
However, studies have shown that: 1) When high-capacity adenoviral vectors carrying λ-phage DNA filler fragments are used in animal experiments, the body generates CTL responses against the λ-phage protein encoded by these fillers, making these vectors immune The system is cleared quickly, and the λ-phage DNA is unstable during the virus production process, which is prone to automatic deletion and recombination
2) Using non-coding regions of the human genome (for example, the intron sequence of the hypoxanthine phosphoribosyltransferase gene containing elements of the matrix attachment region) as a filler can improve the stability of the DNA and will not cause an immune response, but These methods have the potential risk of stuffer-mediated recombination of foreign genes into the human genome

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
  • Complementary type high capacity adenoviral vector and vaccine and gene therapy producers constructed by the same
  • Complementary type high capacity adenoviral vector and vaccine and gene therapy producers constructed by the same
  • Complementary type high capacity adenoviral vector and vaccine and gene therapy producers constructed by the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Construction of complementary high-capacity adenoviral vector I (CHCAd I)

[0058] Elements necessary for adenovirus replication and packaging in CHCAd I include: ITR, packaging signal, E2 gene and late transcription unit. Its acquisition scheme is as follows: the E1 gene and E3 gene have been removed from the first-generation type 5 adenovirus vectors in our laboratory, so the CHCAd I vector can be obtained by removing the E4 gene on this basis. Specifically, the sequences on the left and right sides of the E4 gene to be deleted are first amplified to obtain E4-L and E4-R fragments. These two fragments follow the attached image 3 Inserted into pVAX1 successively in the direction and sequence in order to obtain pVAX1-E4-L-R. EcoRI digested linearized pVAX1-E4-L-R product and SbfI partially digested pAd5 E1-E3- product were transformed into BJ5183 bacteria, and recombined to obtain pAd5E1-E3-E4- (containing the kanamycin resistance gene). If the kanamycin resistance ...

Embodiment 2

[0061] Construction of complementary high-capacity adenoviral vector II (CHCAd II)

[0062] Elements necessary for the replication and packaging of adenovirus in CHCAd II include: ITR, packaging signal and E4 gene. Its acquisition scheme is as follows: from the first generation of adenovirus type 5 vector (from Stratagene's AdEasy TM The E2 gene and the late transcription unit have been removed in the Adenoviral Vector System or other), and only the E4 gene is retained to the CHCAd II vector backbone. Specifically, pAd5 E1-E3 was digested with BstZ17I and HpaI, and the fragments containing the plasmid backbone and E4 gene were recovered and self-ligated to obtain the CHCAd II vector backbone.

[0063] λ-phage genome fragments as filler

[0064] The specific test procedure using λ-bacteriophage genome fragments as fillers is shown in the appendix Figure 4 . Specifically, the sequences on the left and right sides of the selected λ-phage genome fragment are first amplified t...

Embodiment 3

[0074] Optimal selection of λ-bacteriophage genome fragments as fillers

[0075] During the CHCAd II vector construction process, a 23583bp fragment of the adenovirus type 5 genome was deleted, and the average GC content of this fragment was 58%. Use the online software geneGC calculator (http: / / plantst.genomics.purdue.edu / plantst / cgi-bin / geneGC.cgi) to analyze the whole genome of λ-phage, and select the fragment of the λ-phage genome to obtain the 149-21749 interval As a filler, the fragment length is 21601bp, and the average GC content is 567%.

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 a complementary type high-capacity adenoviral vector system and vaccine and gene therapy products constructed by the system. The system includes a pair of complementary adenoviral vectors, and distributes genes which are needed by replication and package of human V type adenovirus in the two vectors that are complementary with each other and cooperate the replication and package of each other. The adenoviral vectors can respectively and jointly carry foreign genes, and the sum of the capacity of the foreign genes on the two vectors can reach 40kb to the utmost extent. The system can be taken as a cell expression vector of mammal, a recombinant vaccine vector and a gene therapy vector, which is applicable to multivalent vaccine carrying multiple foreign genes to prevent the communicable disease and to the gene therapy products carrying a plurality of treatment genes. The invention also selects filling sequences in the complementary virus for preference, thus improving the package efficiency and stability of adenovirus.

Description

1. Technical field [0001] The invention relates to a complementary high-capacity adenoviral vector system and its derivatives. The system includes a pair of complementary adenoviral vectors. The invention also relates to other viral vector systems based on the principle of complementarity. This system can be used as a mammalian cell expression vector, recombinant vaccine and gene therapy vector, and is suitable for multivalent vaccines carrying multiple foreign genes to prevent infectious diseases, and also for gene therapy products carrying multiple therapeutic genes. 2. Background technology [0002] Adenovirus (Adenoviruses, Ad) is a characteristic icosahedral virus capsid, and its genome is a linear double-stranded DNA, about 34kb to 43kb. Ad has a wide host range. Human Ad can be divided into 6 subgroups and about 51 serotypes. Ad can efficiently deliver foreign genes to the nucleus for expression, so the Ad vector system is favored as a mammalian cell expression vec...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861A61K48/00A61K47/46
Inventor 陈凌李锋
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com