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System for rapid production of high-titer and replication-competent adenovirus-free recombinant adenovirus vectors

A recombinant adenovirus, adenovirus technology, applied in the fields of immunology, gene therapy and vaccine technology

Inactive Publication Date: 2008-08-20
德一初C唐 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present invention solves these problems in the art by providing, inter alia, novel adenoviral vectors and methods for producing high titer vaccines by producing RCA-free (replication-competent adenovirus), encoding heterologous nucleic acid in a timely manner For example but not limited to Ad vectors for influenza antigens to achieve

Method used

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  • System for rapid production of high-titer and replication-competent adenovirus-free recombinant adenovirus vectors
  • System for rapid production of high-titer and replication-competent adenovirus-free recombinant adenovirus vectors
  • System for rapid production of high-titer and replication-competent adenovirus-free recombinant adenovirus vectors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0134] Example 1: Generation of Replication Defective Adenovirus Expressing Influenza HA

[0135] Two adenoviral (Ad) vectors encoding influenza HA were constructed using the AdEasy system. The 2 most recent influenza strains [A / Panama / 2007 / 99 (H3N2) and B / Hong Kong / 330 / 01] selected for vaccine production in 2003-2004 were reviewed by The Centers for Disease Control and Prevention. Prevention) (CDC). A / Panama / 2007 / 99 HA was cloned by reverse transcription of influenza RNA followed by amplification of the HA gene by polymerase chain reaction (PCR) using the following primers:

[0136] Table 1: Primer sequences used to amplify influenza genes

[0137]

[0138] These primers contained sequences that anneal to the 5' and 3' ends of the A / Panama / 2007 / 99 HA gene, the eukaryotic ribosome binding site immediately upstream of the HA start ATG codon (Kozak, 1986), and the KpnI site for subsequent cloning. The KpnI fragment containing the full-length HA gene was inserted in the ...

Embodiment 2

[0142] Example 2: Construction of pAdHighα

[0143] Crucell's shuttle plasmid pAdApt was digested separately with restriction enzymes SgrAI+EcoRI, and BstXI+EcoRI. In parallel, the shuttle plasmid pShuttleCMV was digested with SgrAI+BstXI. The resulting pAdApt SgrAI-EcoRI and BstXI-EcoRI fragments were inserted into the SgrAI-BstXI site of pShuttleCMV by 3-route ligation, resulting in a replication-deficient Ad vector. A replication-defective Ad vector encoding the influenza HA gene (Ad Highα PNM2007 / 99.H3) was produced by transfecting the recombinant plasmid into PER.C6 cells. The cytopathic effect (CPE) appeared around 7 days after infection, the same time frame as that required for the AdEasy system in 293 cells (He et al., 1998).

Embodiment 3

[0144] Embodiment 3: Construction of pAdHighβ

[0145] To repair the defective sequence, the CMV promoter, adjacent multiple cloning site, and flanking Ad sequences of pShuttleCMV as 1 unit were replaced by their counterparts from pAdApt by homologous recombination, since the 2 shuttle plasmids share extensively overlapping sequences. However, new markers are also required for selection of recombinants. The full-length tetracycline (Tc) resistance gene from plasmid pBR322 (Backman and Boyer, 1983; Peden, 1983) was performed by PCR using primers 5′-GAGCTCGGTACCTTCTCATGTTTGACAGCTTATCAT-3′ and 5′-TCTAGAGGTACCAACGCTGCCCGAGATGCGCCGCGT-3′ with a built-in KpnI site Amplify. The amplified Tc gene was inserted into the KpnI site of the Amp resistance plasmid pAdApt to generate a new plasmid pAdApt-Tc, which can be selected by application of Amp and Tc to the growth medium.

[0146] The Ad sequence in pShuttleCMV was replaced with its counterpart in pAdApt-Tc using the high efficien...

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Abstract

The present invention relates generally to the fields of gene therapy, immunology, and vaccine technology. More specifically, the invention relates to a novel system that can rapidly generate high titers of adenovirus vectors that are free of replication-competent adenovirus (RCA). Also provided are methods of generating these RCA-free adenoviral vectors, immunogenic or vaccine compositions comprising these RCA-free adenovirus vectors, methods of expressing a heterologous nucleic acid of interest in these adenovirus vectors and methods of eliciting immunogenic responses using these adenovirus vectors.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application Serial No. 60 / 683,638, filed May 23, 2005. [0003] Also mentioned are U.S. Patent Application Serial Nos. 10 / 052,323, filed January 18, 2002; 10 / 116,963, filed April 5, 2002; 10 / 346,021, filed January 16, 2003; and U.S. Patent Nos. 6,706,693; 6,716,823; 6,348,450, and PCT / US / 98 / 16739, filed August 13, 1998. [0004] Each of these applications, patents and documents cited herein, and each of the documents cited in each of these applications, patents and documents (“Application Cited Documents”), and in the Each document referenced or cited in the text or in any document cited in the application during the legal proceeding, and all arguments in support of patentability raised during the legal proceeding thereof, are hereby incorporated herein by reference. field of invention [0005] In general, the present invention relates to the fields of immunology, gen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/64
CPCA61K48/0091A61K2039/543C12N2710/10343A61K2039/5254A61K39/145C12N2710/10351C12N2760/16134A61K2039/5256C12N2760/16122C12N15/86C07K14/005C12N7/00A61K39/12A61P31/16C12N15/09C12N15/861
Inventor D·C·唐J·张K·R·范坎彭
Owner 德一初C唐