Molecular Biotechnology

, Volume 47, Issue 1, pp 43–49 | Cite as

Vector Insert-Targeted Integrative Antisense Expression System for Plasmid Stabilization

  • Jeremy M. Luke
  • Aaron E. Carnes
  • Clague P. Hodgson
  • James A. Williams


Some DNA vaccine and gene therapy vector-encoded transgenes are toxic to the E. coli plasmid production host resulting in poor production yields. For plasmid products undergoing clinical evaluation, sequence modification to eliminate toxicity is undesirable because an altered vector is a new chemical entity. We hypothesized that: (1) insert-encoded toxicity is mediated by unintended expression of a toxic insert-encoded protein from spurious bacterial promoters; and (2) that toxicity could be eliminated with antisense RNA-mediated translation inhibition. We developed the pINT PR PL vector, a chromosomally integrable RNA expression vector, and utilized it to express insert-complementary (anti-insert) RNA from a single defined site in the bacterial chromosome. Anti-insert RNA eliminated leaky fluorescent protein expression from a target plasmid. A toxic retroviral gag pol helper plasmid produced in a gag pol anti-insert strain had fourfold improved plasmid fermentation yields. Plasmid fermentation yields were also fourfold improved when a DNA vaccine plasmid containing a toxic Influenza serotype H1 hemagglutinin transgene was grown in an H1 sense strand anti-insert production strain, suggesting that in this case toxicity was mediated by an antisense alternative reading frame-encoded peptide. This anti-insert chromosomal RNA expression technology is a general approach to improve production yields with plasmid-based vectors that encode toxic transgenes, or toxic alternative frame peptides.


Plasmid DNA vaccine Antisense RNA Non-viral vector Vector stabilization Strain engineering 



We thank Sheryl Anderson and Angela Schukar for cleaning, batching, and operating fermentors. This article described work that was supported by the National Institute of Health [R44GM072141 and R43GM073394 to J.A.W.].

Conflicts of Interest Statement

JML, AEC, CPH and JAW have an equity interest in Nature Technology Corporation.


  1. 1.
    Kutzler, M. A., & Weiner, D. B. (2008). DNA vaccines: Ready for prime time? Nature Reviews Genetics, 9, 776–788.CrossRefGoogle Scholar
  2. 2.
    Williams, J. A., Carnes, A. E., & Hodgson, C. P. (2009). Plasmid DNA vector design; impact on efficacy, safety and upstream production. Biotechnology Advances, 27, 353–370.CrossRefGoogle Scholar
  3. 3.
    Mairhofer, J., & Grabherr, R. (2008). Rational vector design for efficient non-viral gene delivery: Challenges facing the use of plasmid DNA. Molecular Biotechnology, 39, 97–104.CrossRefGoogle Scholar
  4. 4.
    Engels, P., & Meyer, P. (1993). Inactivation of the transcriptional-dependent inhibition of plasmid replication: a selection method for cloning large DNA fragments. Biotechniques, 14, 324–325.Google Scholar
  5. 5.
    Brosius, J. (1984). Toxicity of an overproduced foreign gene product in Escherichia coli and its use in plasmid vectors for the selection of transcription terminators. Gene, 27, 161–172.CrossRefGoogle Scholar
  6. 6.
    Chen, W., Kallio, P. T., & Bailey, J. E. (1993). Construction and characterization of a novel cross-regulation system for regulating cloned gene expression in Escherichia coli. Gene, 130, 15–22.CrossRefGoogle Scholar
  7. 7.
    Chen, J. D., & Morrision, D. A. (1987). Cloning of Streptococcus pneumoniae DNA fragments in Escherichia coli requires vectors protected by strong transcriptional terminators. Gene, 55, 179–187.CrossRefGoogle Scholar
  8. 8.
    Cooke, J. R., McKie, E. A., Ward, J. M., & Keshavarz-Moore, E. (2004). Impact of intrinsic DNA structure on processing of plasmids for gene therapy and DNA vaccines. Journal of Biotechnology, 114, 239–254.CrossRefGoogle Scholar
  9. 9.
    Weiner, D. B., Zhang, D., & Cohen, A. (2005). Expression system for cloning toxic genes. United States Patent 6881558.Google Scholar
  10. 10.
    Saida, F., Uzan, M., Odaert, B., & Bontems, F. (2006). Expression of highly toxic genes in E. coli: Special strategies and genetic tools. Current Protein and Peptide Science, 7, 47–56.CrossRefGoogle Scholar
  11. 11.
    Boyd, A. C., Popp, F., Michaelis, U., Davidson, H., Davidson-Smith, H., Doherty, A., et al. (1999). Insertion of natural intron 6a–6b into a human cDNA-derived gene therapy vector for cystic fibrosis improves plasmid stability and permits facile RNA/DNA discrimination. Journal of Gene Medicine, 1, 312–321.CrossRefGoogle Scholar
  12. 12.
    Futterer, J., Gordon, K., Pfeiffer, P., & Hohn, T. (1988). The instability of a recombinant plasmid, caused by a prokaryotic-like promoter within the eukaryotic insert, can be alleviated by expression of antisense RNA. Gene, 67, 141–145.CrossRefGoogle Scholar
  13. 13.
    Haldimann, A., & Wanner, B. L. (2001). Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria. Journal of Bacteriology, 183, 6384–6393.CrossRefGoogle Scholar
  14. 14.
    Williams, J. A., Luke, J., Langtry, S., Anderson, S., Hodgson, C. P., & Carnes, A. E. (2009). Generic plasmid DNA production platform incorporating low metabolic burden seed-stock and fed-batch fermentation processes. Biotechnology and Bioengineering, 103, 1129–1143.CrossRefGoogle Scholar
  15. 15.
    Williams, J. A. (2008). Vectors and methods for genetic immunization. WO 2008/153733.Google Scholar
  16. 16.
    Carnes, A. E., Hodgson, C. P., Luke, J., Vincent, J., & Williams, J. A. (2009). Plasmid DNA production combining antibiotic-free selection, inducible high yield fermentation, and novel autolytic purification. Biotechnology and Bioengineering, 104, 505–515.CrossRefGoogle Scholar
  17. 17.
    Kemmer, C., & Neubauer, P. (2006). Antisense RNA based down-regulation of RNase E in E. coli. Microbial Cell Factories, 5, 38.CrossRefGoogle Scholar
  18. 18.
    Luke, J., Carnes, A. E., Hodgson, C. P., & Williams, J. A. (2009). Improved antibiotic-free DNA vaccine vectors utilizing a novel RNA based plasmid selection system. Vaccine, 27, 6454–6459.CrossRefGoogle Scholar
  19. 19.
    Datsenko, K. A., & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences USA, 97, 6640–6645.CrossRefGoogle Scholar
  20. 20.
    Makrides, S. C. (1996). Strategies for achieving high-level expression of genes in Escherichia coli. Microbiological Reviews, 60, 512–538.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Jeremy M. Luke
    • 1
  • Aaron E. Carnes
    • 1
  • Clague P. Hodgson
    • 1
  • James A. Williams
    • 1
  1. 1.Nature Technology CorporationLincolnUSA

Personalised recommendations