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Journal of Virology, November 2002, p. 10734-10744, Vol. 76, No. 21
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.21.10734-10744.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Efficient Generation and Amplification of High-Capacity Adeno-Associated Virus/Adenovirus Hybrid Vectors

Manuel A. F. V. Gonçalves,1* Ietje van der Velde,1 Josephine M. Janssen,1 Bram T. H. Maassen,1 Evert H. Heemskerk,2 Dirk-Jan E. Opstelten,2 Shoshan Knaän-Shanzer,1 Dinko Valerio,1 and Antoine A. F. de Vries1*

Gene Therapy Section, Department of Molecular Cell Biology, Leiden University Medical Center,1 Crucell N.V., Leiden, The Netherlands2

Received 17 April 2002/ Accepted 26 July 2002

Effective gene therapy is dependent on safe gene delivery vehicles that can achieve efficient transduction and sustained transgene expression. We are developing a hybrid viral vector system that combines in a single particle the large cloning capacity and efficient cell cycle-independent nuclear gene delivery of adenovirus (Ad) vectors with the long-term transgene expression and lack of viral genes of adeno-associated virus (AAV) vectors. The strategy being pursued relies on coupling the AAV DNA replication mechanism to the Ad encapsidation process through packaging of AAV-dependent replicative intermediates provided with Ad packaging elements into Ad capsids. The generation of these high-capacity AAV/Ad hybrid vectors takes place in Ad early region 1 (E1)-expressing cells and requires an Ad vector with E1 deleted to complement in trans both AAV helper functions and Ad structural proteins. The dependence on a replicating helper Ad vector leads to the contamination of AAV/Ad hybrid vector preparations with a large excess of helper Ad particles. This renders the further propagation and ultimate use of these gene delivery vehicles very difficult. Here, we show that Cre/loxP-mediated genetic selection against the packaging of helper Ad DNA can reduce helper Ad vector contamination by 99.98% without compromising hybrid vector rescue. This allowed amplification of high-capacity AAV/Ad hybrid vectors to high titers in a single round of propagation.

* Corresponding author. Mailing address: Gene Therapy Section, Department of Molecular Cell Biology, Leiden University Medical Center (LUMC), Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. Phone: 31 71 5271971 or 5271998. Fax: 31 71 5276180. E-mail: m.goncalves{at}lumc.nl or a.a.f.de_vries{at}lumc.nl.

Journal of Virology, November 2002, p. 10734-10744, Vol. 76, No. 21
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.21.10734-10744.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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