DNA Nuclear Targeting Sequences for Non-Viral Gene Delivery

• Published in: Pharmaceutical research Vol. 28; no. 7; pp. 1707 - 1722
• Main Authors: van Gaal, Ethlinn V. B., Oosting, Ronald S., van Eijk, Roel, Bakowska, Marta, Feyen, Dries, Kok, Robbert Jan, Hennink, Wim E., Crommelin, Daan J. A., Mastrobattista, Enrico
• Format: Journal Article
• Language: English
• Published: Boston Springer US 01.07.2011; Springer; Springer Nature B.V
• Subjects: Base Sequence; Biochemistry; Biological and medical sciences; Biomedical and Life Sciences; Biomedical Engineering and Bioengineering; Biomedicine; Cell Line, Tumor; Cell Nucleus - genetics; Deoxyribonucleic acid; DNA; Electroporation; Flow Cytometry; Gene expression; Gene therapy; Gene Transfer Techniques; General pharmacology; Genetic Vectors - genetics; HeLa Cells; Humans; Medical Law; Medical sciences; Molecular Sequence Data; Pharmaceutical sciences; Pharmaceutical technology. Pharmaceutical industry; Pharmacology. Drug treatments; Pharmacology/Toxicology; Pharmacy; Plasmids; Plasmids - genetics; Polymerase Chain Reaction; Research Paper; non-viral gene delivery; DNA nuclear Targeting Sequences; nuclear import; DNA; Polyelectrolyte; Target; Pharmaceutical technology; Targeting; Nucleotide sequence; Gene therapy; Nucleic acid; Genetic transfer
• ISSN: 0724-8741; 1573-904X; 1573-904X
• DOI: 10.1007/s11095-011-0407-8

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.