Wilhelm bernhard workshop on the cell nucleus

Enhancement of nuclear uptake of therapeutic genes and macro-molecules by using NLS and PTD containing viral peptides

Sanders N, Vandenbroucke R, De Smedt S, Vanderleyden E, Lucas B, Remaut K Demeester J

Laboratory of General Biochemistry and Physical Pharmacy, Gent University, Gent, Belgium
Gene therapy is a novel approach to treat genetic based diseases by bringing therapeutic genes or oligonucleotides inside the nucleus or cytoplasm of target cells. Two types of DNA carriers are currently used: viral and non-viral. Because of the importance of safety in clinical applications, much attention has been given to non-viral carriers (e.g. cationic polymers and liposomes). However, these gene carriers are less effective in vivo. Indeed, it is widely recognized that in vivo many biological barriers complicate the delivery of exogenous pDNA to the cell nucleus. At the moment, the nuclear membrane is considered as a major barrier for pDNA. Therefore, we evaluated whether attachment of HIV-1 TAT (37-72) and other NLS containing peptides to fluorescent labeled pDNA and BSA can cause nuclear localization of these macromolecules. After manufacturing Cy5-pDNA and FITC-BSA with and without NLS peptide we microinjected these constructs, into Vero cells and followed, using a confocal scanning laser microscope, the fluorescence enhancement in the nucleus. As an internal control high molecular weight fluorescent, labeled dextrans were co-injected into the cells. When the nuclear membrane is intact, it is known that these dextrans are excluded from the nucleus. Micro-injected pDNA or BSA did not locate in the nucleus after 20 min. NLS conjugated to pDNA by electrostatic interaction did not cause nuclear localization of the pDNA. However, covalent linking of TAT to BSA resulted into a very rapid nuclear localization after micro-injection. These observations indicate that a covalent linking of an NLS petides to its cargo may, probably, be a requisite for an efficient nuclear localization. Moreover, we also observed that FITC-BSA covalently coupled with TAT was able to cross the cell membrane and the nuclear membrane in a few minutes. In further experiments we want to evaluate whether covalently linked NLS peptides to pDNA can cause a nuclear localization of the pDNA.