Increase in the titer of lentiviral vectors expressing potassium channels by current blockade during viral vector production.

Masayoshi Okada,Naaz Andharia,Hiroko Matsuda

doi:10.1186/s12868-015-0159-1

Masayoshi Okada, Naaz Andharia + Show 1 more

Open Access

https://doi.org/10.1186/s12868-015-0159-1

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Journal: BMC neuroscience	Publication Date: May 5, 2015
Citations: 37	License type: CC BY 4.0

Affiliation: Kansai Medical University

Abstract

BackgroundHigh titers of lentiviral vectors are required for the efficient transduction of a gene of interest. During preparation of lentiviral the vectors, the protein of interest is inevitably expressed in the viral vector-producing cells. This expression may affect the production of the lentiviral vector.MethodsWe prepared lentiviral vectors expressing inwardly rectifying potassium channel (Lv-Kir2.1), its dominant-negative form (Lv-Kir-DN), and other K+ channels, using the ubiquitously active β-actin and neuron-specific synapsin I promoters.ResultsThe titer of Lv-Kir-DN was higher than that of Lv-Kir2.1, suggesting a negative effect of induced K+ currents on viral titer. We then blocked Kir2.1 currents with the selective blocker Ba2+ during Lv-Kir2.1 production, and obtained about a 5-fold increase in the titer. Higher extracellular K+ concentrations increased the titer of Lv-Kir2.1 about 9-fold. With a synapsin I promoter Ba2+ increased the titer because of the moderate expression of Kir2.1 channel. Channel blockade also increased the titers of the lentivirus expressing Kv1.4 and TREK channels, but not HERG. The increase in titer correlated with the K+ currents generated by the channels expressed.ConclusionIn the production of lentivirus expressing K+ channels, titers are increased by blocking K+ currents in the virus-producing cells. This identifies a crucial issue in the production of viruses expressing membrane channels, and should facilitate basic and gene therapeutic research on channelopathies.

Highlights

High titers of lentiviral vectors are required for the efficient transduction of a gene of interest
Because the K+ current is the major determinant of membrane potential, these results consistently suggest that the viral-vector-producing ability is reduced by an excessive K+ current
The titers and membrane potentials of Kv1.4 and TREK differed in their ranking, which is probably attributable to the incomplete blockade of the TREK channel by BaCl2

Summary

Introduction

High titers of lentiviral vectors are required for the efficient transduction of a gene of interest. During preparation of lentiviral the vectors, the protein of interest is inevitably expressed in the viral vector-producing cells. This expression may affect the production of the lentiviral vector. Especially lentiviral vectors, are useful in transducing genes of interest in vitro, in situ, and in vivo. Lentiviral vectors are promising tools for gene therapy because they can transduce postmitotic cells, allow long-lasting expression, and exhibit low toxicity and low oncogenic activity [1]. High-titer lentiviral vectors are required for efficient transduction and timeand cost-savings. Because most lentiviral vectors contain ubiquitously active promoters, the proteins of interest are inevitably expressed in the viral-. The loss-offunction mutation of strongly inwardly rectifying K+

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