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Journal of Virology, August 2006, p. 8100-8113, Vol. 80, No. 16
0022-538X/06/$08.00+0     doi:10.1128/JVI.00687-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Transposon-Assisted Cloning and Traceless Mutagenesis of Adenoviruses: Development of a Novel Vector Based on Species D{dagger}

Zsolt Ruzsics,2 Markus Wagner,2,{ddagger} Andrea Osterlehner,2,§ Jonathan Cook,1 Ulrich Koszinowski,2 and Hans-Gerhard Burgert1,2*

University of Warwick, Department of Biological Sciences, Coventry CV4 7AL, United Kingdom,1 Max-von-Pettenkofer Institute, Gene Center, Ludwig-Maximilians-University, 81377 Munich, Germany2

Received 5 April 2005/ Accepted 31 May 2006

Until recently, adenovirus (Ad)-mediated gene therapy was almost exclusively based on human Ad type 5 (Ad5). Preexisting immunity and the limited, coxsackievirus and adenovirus receptor-dependent tropism of Ad5 stimulated attempts to exploit the natural diversity in tropism of the other 50 known human Ad serotypes. Aiming in particular at immunotherapy and vaccination, we have screened representative serotypes from different Ad species for their ability to infect dendritic cells. Ad19a, an Ad from species D, was selected for development as a new vector for vaccination and cancer gene therapy. To clone and manipulate its genome, we have developed a novel methodology, coined "exposon mutagenesis," that allows the rapid and precise introduction of virtually any genetic alteration (deletions, point mutations, or insertions) into recombinant Ad bacterial artificial chromosomes. The versatility of the system was exemplified by deleting the E3 region of Ad19a, by specifically knocking out expression of a species-specific E3 gene, E3/49K, and by reinserting E3/49K into an E3 null Ad19a mutant. The technology requires only limited sequence information and is applicable to other Ad species. Therefore, it should be extremely valuable for the analysis of gene functions from any Ad species. In addition, a basic, replication-defective E1- and E3-deleted Ad19a vector expressing GFP (Ad19aGFP) was generated. This new vector based on species D Ads exhibits a very promising tropism for lymphoid and muscle cells and shows great potential as an alternative vector for transduction of cell types that are resistant to or only poorly transduced by conventional Ad5-based vectors.

* Corresponding author. Mailing address: Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom. Phone: 44-2476-524744. Fax: 44-2476-523568. E-mail: H-G.Burgert{at}warwick.ac.uk.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org.

{ddagger} Present address: Bavaria Nordic GmbH, Fraunhoferstr. 13, 82152 Martinsried, Germany.

§ Present address: Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany.

Journal of Virology, August 2006, p. 8100-8113, Vol. 80, No. 16
0022-538X/06/$08.00+0     doi:10.1128/JVI.00687-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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