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Journal of Virology, February 2006, p. 1672-1679, Vol. 80, No. 4
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.4.1672-1679.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Efficient Site-Specific Integration of Large Transgenes by an Enhanced Herpes Simplex Virus/Adeno-Associated Virus Hybrid Amplicon Vector

Qiang Liu, Claudio F. Perez, and Yaming Wang^*

Department of Anesthesia, Brigham & Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

Received 25 August 2005/ Accepted 18 November 2005

We previously demonstrated that a herpes simplex virus type 1 (HSV-1)/adeno-associated virus (AAV) hybrid amplicon vector constructed by inserting the sequences of regulatory protein (rep) and inverted terminal repeats of AAV into an HSV amplicon vector resulted in the enhanced stability of transgene expression compared to the original HSV-1 amplicon vector. However, problems related to the expression of Rep compromised its therapeutic applications. We report here a new HSV/AAV hybrid amplicon vector system that not only solved problems associated with Rep expression but also markedly improved the stable transduction efficiency of this vector. This new HSV/AAV vector is designed in a way that little or no Rep would be expressed in packaging cells, but it can be expressed in transduced cells if Cre recombinase is provided. Furthermore, Rep expression will be automatically suppressed as a consequence of Rep-mediated integration. Our results showed that the new hybrid amplicon vector yielded titers comparable to those of standard amplicon vectors. When Cre-expressing 293 cells were transduced, a low level of Rep expression was detected, and stable transduction was achieved in 22% of transduced cells; of those cells, 70% transduction was achieved by Rep-mediated site-specific integration. In the majority of the stably transduced cells, Rep expression was no longer observed. Our results also proved that this vector system is capable of efficiently accommodating and site-specifically integrating large transgenes, such as the full-length dystrophin expression cassette. Thus, the new HSV/AAV vector demonstrated unique advantages in safe and effective delivery of long-lasting transgene expression into human cells.

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* Corresponding author. Mailing address: Department of Anesthesia, Brigham & Women's Hospital, 75 Francis Street, SR 153, Boston, MA 02115. Phone: (617) 732-6879. Fax: (617) 732-6927. E-mail: yaming{at}zeus.bwh.harvard.edu.

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Journal of Virology, February 2006, p. 1672-1679, Vol. 80, No. 4
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.4.1672-1679.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Murphy, M., Gomos-Klein, J., Stankic, M., Falck-Pedersen, E. (2007). Adeno-Associated Virus Type 2 p5 Promoter: a Rep-Regulated DNA Switch Element Functioning in Transcription, Replication, and Site-Specific Integration. J. Virol. 81: 3721-3730 [Abstract] [Full Text]  
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