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Journal of Virology, April 2006, p. 3293-3300, Vol. 80, No. 7
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.7.3293-3300.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Plasmid DNA Sequences Present in Conventional Herpes Simplex Virus Amplicon Vectors Cause Rapid Transgene Silencing by Forming Inactive Chromatin

Masataka Suzuki, Kazue Kasai, and Yoshinaga Saeki*

Dardinger Laboratory for Neuro-oncology and Neurosciences, Department of Neurological Surgery, The Ohio State University Medical Center, Columbus, Ohio

Received 21 October 2005/ Accepted 17 January 2006

The herpes simplex virus (HSV)-based amplicon vector, a bacterial-viral-mammalian cell shuttle system, holds promise as a versatile gene delivery vehicle because of its large transgene capacity. However, amplicon-mediated transgene expression is often transient. We hypothesized that the presence of prokaryotic DNA sequences within the packaged vector genome can trigger transcriptional silencing of the entire vector sequence. To test this, we constructed a novel amplicon vector devoid of bacterial sequences (minicircle [MC] amplicon). Although the same dose of the minicircle amplicon vector in normal human fibroblasts resulted in an expression of luciferase approximately 20 times higher than that caused by the conventional amplicon vector, no significant difference was observed in copy numbers of luciferase DNA between MC amplicon- and control-transduced cells. Quantitative analyses of levels of luciferase mRNA revealed that differential expression of luciferase was controlled at the transcriptional level. Chromatin immunoprecipitation PCR analyses of several regions of vector genomes revealed that the bacterial sequences found in the conventional amplicon DNA were associated with an inactive form of chromatin immediately after infection. The presence of bacterial sequences also affected the remaining vector sequences in the conventional amplicon vector. Finally, nude mice injected with the MC amplicon exhibited higher and more sustained expression of luciferase than those injected with the conventional amplicon, confirming the usefulness of the MC amplicon devoid of bacterial sequences. Although additional improvements are absolutely required, these findings are a significant first step toward developing a novel HSV amplicon vector that can achieve enhanced long-term transgene expression.

* Corresponding author. Mailing address: Dardinger Laboratory for Neuro-oncology and Neurosciences, Department of Neurological Surgery, The Ohio State University Medical Center, 385B Wiseman Hall, 400 West 12th Avenue, Columbus, OH 43210. Phone: (614) 292-5530. Fax: (614) 688-4882. E-mail: saeki.6{at}osu.edu.

Journal of Virology, April 2006, p. 3293-3300, Vol. 80, No. 7
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.7.3293-3300.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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