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Mol Ther Methods Clin Dev. 2015 Mar 18;2:15004. doi: 10.1038/mtm.2015.4. eCollection 2015.

High-level production of replication-defective human immunodeficiency type 1 virus vector particles using helper-dependent adenovirus vectors.

Author information

1
Division of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine , New Haven, Connecticut, USA.
2
Department of Molecular and Human Genetics, Baylor College of Medicine , Houston, Texas, USA.

Abstract

Gene transfer vectors based upon human immunodeficiency virus type 1 (HIV) are widely used in bench research applications and increasingly in clinical investigations, both to introduce novel genes but also to reduce expression of unwanted genes of the host and pathogen. At present, the vast majority of HIV-based vector supernatants are produced in 293T cells by cotransfection of up to five DNA plasmids, which is subject to variability and difficult to scale. Here we report the development of a HIV-based vector production system that utilizes helper-dependent adenovirus (HDAd). All necessary HIV vector components were inserted into one or more HDAds, which were then amplified to very high titers of ~10(13) vp/ml. These were then used to transduce 293-based cells to produce HIV-based vector supernatants, and resultant VSV G-pseudotyped lentiviral vector (LV) titers and total IU were 10- to 30-fold higher, compared to plasmid transfection. Optimization of HIV-based vector production depended upon maximizing expression of all HIV vector components from HDAd. Supernatants contained trace amounts of HDAd but were free of replication-competent lentivirus. This production method should be applicable to other retroviral vector systems. Scalable production of HIV-based vectors using this two-step procedure should facilitate their clinical advancement.

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