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Journal of Virology, December 1999, p. 10426-10439, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Single-Step Conversion of Cells to Retrovirus Vector Producers with Herpes Simplex Virus-Epstein-Barr Virus Hybrid Amplicons

Miguel Sena-Esteves,1,2 Yoshinaga Saeki,3 Sara M. Camp,1,2 E. Antonio Chiocca,3 and Xandra O. Breakefield1,2,*

Molecular Neurogenetics Unit,1 Department of Neurology,2 and Department of Neurosurgery,3 Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129

Received 23 March 1999/Accepted 25 August 1999

We report here on the development and characterization of a novel herpes simplex virus type 1 (HSV-1) amplicon-based vector system which takes advantage of the host range and retention properties of HSV-Epstein-Barr virus (EBV) hybrid amplicons to efficiently convert cells to retrovirus vector producer cells after single-step transduction. The retrovirus genes gag-pol and env (GPE) and retroviral vector sequences were modified to minimize sequence overlap and cloned into an HSV-EBV hybrid amplicon. Retrovirus expression cassettes were used to generate the HSV-EBV-retrovirus hybrid vectors, HERE and HERA, which code for the ecotropic and the amphotropic envelopes, respectively. Retrovirus vector sequences encoding lacZ were cloned downstream from the GPE expression unit. Transfection of 293T/17 cells with amplicon plasmids yielded retrovirus titers between 106 and 107 transducing units/ml, while infection of the same cells with amplicon vectors generated maximum titers 1 order of magnitude lower. Retrovirus titers were dependent on the extent of transduction by amplicon vectors for the same cell line, but different cell lines displayed varying capacities to produce retrovirus vectors even at the same transduction efficiencies. Infection of human and dog primary gliomas with this system resulted in the production of retrovirus vectors for more than 1 week and the long-term retention and increase in transgene activity over time in these cell populations. Although the efficiency of this system still has to be determined in vivo, many applications are foreseeable for this approach to gene delivery.

* Corresponding author. Mailing address: Molecular Neurogenetics Unit, Department of Neurology, Massachusetts General Hospital, CNY 6205, Bldg. 149, 13th St., Charlestown, MA 02129. Phone: (617) 726-5728. Fax: (617) 724-1537. E-mail: breakefield{at}helix.mgh.harvard.edu

Journal of Virology, December 1999, p. 10426-10439, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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