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Journal of Virology, January 2000, p. 281-294, Vol. 74, No. 1
0022-538X/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Conditional Site-Specific Integration into Human Chromosome 19 by Using a Ligand-Dependent Chimeric Adeno-Associated Virus/Rep Protein

Daniela Rinaudo, Stefania Lamartina, Giuseppe Roscilli, Gennaro Ciliberto, and Carlo Toniatti*

Department of Genetics, Istituto di Ricerche di Biologia Molecolare, I.R.B.M.---Piero Angeletti, 00040 Pomezia (Rome), Italy

Received 7 May 1999/Accepted 21 September 1999

It is of great interest for gene therapy to develop vectors that drive the insertion of a therapeutic gene into a chosen specific site on the cellular genome. Adeno-associated virus (AAV) is unique among mammalian viruses in that it integrates into a distinct region of human chromosome 19 (integration site AAVS1). The inverted terminal repeats (ITRs) flanking the AAV genome and the AAV-encoded nonstructural proteins Rep78 and/or Rep68 are the only viral elements necessary and sufficient for site-specific integration. However, it is also known that unrestrained Rep activity may cause nonspecific genomic rearrangements at AAVS1 and/or have detrimental effects on cell physiology. In this paper we describe the generation of a ligand-dependent form of Rep, obtained by fusing a C-terminally deleted Rep68 with a truncated form of the hormone binding domain of the human progesterone receptor, which does not bind progesterone but binds only its synthetic antagonist RU486. The activity of this chimeric protein, named Rep1-491/P, is highly dependent on RU486 in various assays: in particular, it triggers site-specific integration at AAVS1 of an ITR-flanked cassette in a ligand-dependent manner, as efficiently as wild-type Rep68 but without generating unwanted genomic rearrangement at AAVS1.

* Corresponding author. Mailing address: Istituto di Ricerche di Biologia Molecolare, I.R.B.M.---P. Angeletti, Via Pontina Km 30.600, 00040 Pomezia (Rome), Italy. Phone: 39-06-91093668. Fax: 39-06-91093654. E-mail: toniatti{at}irbm.it.

Journal of Virology, January 2000, p. 281-294, Vol. 74, No. 1
0022-538X/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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