Publication

DNA nuclear targeting sequences for non-viral gene delivery

van Gaal, E. V. B., Oosting, R. S., van Eijk, R., Bakowska, M., Feyen, D., Kok, R. J., Hennink, W. E., Crommelin, D. J. A. & Mastrobattista, E., Jul-2011, In : Pharmaceutical Research. 28, 7, p. 1707-22 16 p.

Research output: Contribution to journalArticleAcademicpeer-review

  • Ethlinn V B van Gaal
  • Ronald S Oosting
  • Roel van Eijk
  • Marta Bakowska
  • Dries Feyen
  • Robbert Jan Kok
  • Wim E Hennink
  • Daan J A Crommelin
  • Enrico Mastrobattista

PURPOSE: To evaluate if introduction of DNA nuclear Targeting Sequences (DTS; i.e. recognition sequences for endogenous DNA-binding proteins) in plasmid DNA (pDNA) leads to increased transfection efficiency of non-viral gene delivery by virtue of enhanced nuclear import of the pDNA.

METHODS: A set of DTS was identified and cloned into EGFP-reporter plasmids controlled by the CMV-promoter. These pDNA constructs were delivered into A431 and HeLa cells using standard electroporation, pEI-based polyfection or lipofection methods. The amount of pDNA delivered into the nucleus was determined by qPCR; transfection efficiency was determined by flow cytometry.

RESULTS: Neither of these DTS increased transgene expression. We varied several parameters (mitotic activity, applied dose and delivery strategy), but without effect. Although upregulated transgene expression was observed after stimulation with TNF-α, this effect could be ascribed to non-specific upregulation of transcription rather than enhanced nuclear import. Nuclear copy numbers of plasmids containing or lacking a DTS did not differ significantly after lipofectamine-based transfection in dividing and non-dividing cells.

CONCLUSION: No beneficial effects of DTS on gene expression or nuclear uptake were observed in this study.

Original languageEnglish
Pages (from-to)1707-22
Number of pages16
JournalPharmaceutical Research
Volume28
Issue number7
Publication statusPublished - Jul-2011
Externally publishedYes

    Keywords

  • Base Sequence, Cell Line, Tumor, Cell Nucleus, Electroporation, Flow Cytometry, Gene Transfer Techniques, Genetic Vectors, HeLa Cells, Humans, Molecular Sequence Data, Plasmids, Polymerase Chain Reaction

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