caa a22 4500 445312394 CHVBK 20180317142634.0 cr unu---uuuuu 170323e20110701xx s 000 0 eng 10.1007/s11095-011-0407-8 doi (NATIONALLICENCE)springer-10.1007/s11095-011-0407-8 DNA Nuclear Targeting Sequences for Non-Viral Gene Delivery [Elektronische Daten] [Ethlinn van Gaal, Ronald Oosting, Roel van Eijk, Marta Bakowska, Dries Feyen, Robbert Kok, Wim Hennink, Daan Crommelin, Enrico Mastrobattista] ABSTRACT: 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. The Author(s), 2011 DNA nationallicence DNA nuclear Targeting Sequences nationallicence non-viral gene delivery nationallicence nuclear import nationallicence van Gaal Ethlinn Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA, Utrecht, The Netherlands aut Oosting Ronald Department of Pharmacology, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA, Utrecht, The Netherlands aut van Eijk Roel Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA, Utrecht, The Netherlands aut Bakowska Marta Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA, Utrecht, The Netherlands aut Feyen Dries Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA, Utrecht, The Netherlands aut Kok Robbert Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA, Utrecht, The Netherlands aut Hennink Wim Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA, Utrecht, The Netherlands aut Crommelin Daan Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA, Utrecht, The Netherlands aut Mastrobattista Enrico Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA, Utrecht, The Netherlands aut Pharmaceutical Research Springer US; http://www.springer-ny.com 28/7(2011-07-01), 1707-1722 0724-8741 28:7<1707 2011 28 11095 https://doi.org/10.1007/s11095-011-0407-8 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s11095-011-0407-8 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- van Gaal Ethlinn Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA, Utrecht, The Netherlands aut NATIONALLICENCE 700 1- Oosting Ronald Department of Pharmacology, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA, Utrecht, The Netherlands aut NATIONALLICENCE 700 1- van Eijk Roel Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA, Utrecht, The Netherlands aut NATIONALLICENCE 700 1- Bakowska Marta Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA, Utrecht, The Netherlands aut NATIONALLICENCE 700 1- Feyen Dries Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA, Utrecht, The Netherlands aut NATIONALLICENCE 700 1- Kok Robbert Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA, Utrecht, The Netherlands aut NATIONALLICENCE 700 1- Hennink Wim Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA, Utrecht, The Netherlands aut NATIONALLICENCE 700 1- Crommelin Daan Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA, Utrecht, The Netherlands aut NATIONALLICENCE 700 1- Mastrobattista Enrico Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA, Utrecht, The Netherlands aut NATIONALLICENCE 773 0- Pharmaceutical Research Springer US; http://www.springer-ny.com 28/7(2011-07-01), 1707-1722 0724-8741 28:7<1707 2011 28 11095 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer