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Improved antibiotic-free plasmid vector design by incorporation of transient expression enhancers

Gene Therapy volume 18, pages 334–343 (2011)Cite this article

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Abstract

Methods to improve plasmid-mediated transgene expression are needed for gene medicine and gene vaccination applications. To maintain a low risk of insertional mutagenesis-mediated gene activation, expression-augmenting sequences would ideally function to improve transgene expression from transiently transfected intact plasmid, but not from spurious genomically integrated vectors. We report herein the development of potent minimal, antibiotic-free, high-manufacturing-yield mammalian expression vectors incorporating rationally designed additive combinations of expression enhancers. The SV40 72 bp enhancer incorporated upstream of the cytomegalovirus (CMV) enhancer selectively improved extrachromosomal transgene expression. The human T-lymphotropic virus type I (HTLV-I) R region, incorporated downstream of the CMV promoter, dramatically increased mRNA translation efficiency, but not overall mRNA levels, after transient transfection. A similar mRNA translation efficiency increase was observed with plasmid vectors incorporating and expressing the protein kinase R-inhibiting adenoviral viral associated (VA)1 RNA. Strikingly, HTLV-I R and VA1 did not increase transgene expression or mRNA translation efficiency from plasmid DNA after genomic integration. The vector platform, when combined with electroporation delivery, further increased transgene expression and improved HIV-1 gp120 DNA vaccine-induced neutralizing antibody titers in rabbits. These antibiotic-free vectors incorporating transient expression enhancers are safer, more potent alternatives to improve transgene expression for DNA therapy or vaccination.

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Acknowledgements

We thank Marni England-Hill (Aldevron) and Jennifer Bath (Concordia College) for oversight of the rabbit study at Aldevron, and Danielle Shea (University of Nebraska, Lincoln) for performing flow cytometry. We also thank Kim Hanson (Nature Technology) for purifying the plasmid DNA used in this study and Sheryl Anderson (Nature Technology) for linear vector preparation. This paper described work supported by NIH grants R44 GM072141-03 and R43 GM080768-01 to JAW. AML is supported by a Specialized Centers for Cell-based Therapy Grant NIH-NHLBI 1 U54 HL081007 and an Amy Strelzer Manasevit Scholar Award. UG is supported by an Asbmt Young Investigator Award and a Leukemia and Lymphoma Society Special Fellow in Clinical Research Award.

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Author notes

  1. S X Du

    Present address: 4Current address: Simcere Pharmaceutical Group, Nanjing, 210042, PR China.,

  2. R G Whalen

    Present address: 5Current address: Altravax Inc., 5252 Del Rey Avenue, Suite B, Sunnyvale, CA 94085, USA.,

Authors and Affiliations

  1. Nature Technology Corporation, Lincoln, NE, USA

    J M Luke, J M Vincent, C P Hodgson & J A Williams

  2. Maxygen Inc., Redwood City, CA, USA

    S X Du & R G Whalen

  3. Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA

    U Gerdemann & A M Leen

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  1. J M Luke
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Correspondence to J A Williams.

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Competing interests

JML, JMV, CPH and JAW have an equity interest in Nature Technology Corporation. SXD and RGW have an equity interest in AltraVax.

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Luke, J., Vincent, J., Du, S. et al. Improved antibiotic-free plasmid vector design by incorporation of transient expression enhancers. Gene Ther 18, 334–343 (2011). https://doi.org/10.1038/gt.2010.149

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