Regis Cartier

Utilization of synthetic peptides containing nuclear localization signals for nonviral gene transfer systems

The ability of nonviral gene delivery systems to overcome extracellular and intracellular barriers is a critical issue for future clinical applications. In recent years, several efforts were focused on the elucidation of the gene transfer mechanisms and on the development of multicomponent systems in order to improve both targeted gene delivery and transfection efficiency. The transport of the therapeutic DNA from the cytoplasm into the nucleus is an inefficient process and is considered as the major limiting step in nondividing cells. One of the strategies to improve nuclear uptake of DNA is taking advantage of the cellular nuclear import machinery. Synthetic peptides containing a nuclear localization signal (NLS) are bound to the DNA so that the resulting DNA–NLS complex can be recognized as a nuclear import substrate by specific intracellular receptor proteins. In this review, we critically summarize recent studies applying this approach with a particular focus on NLS-sequence specificity. Implications of the observed results are also discussed in regards to future developments of this technology.

Polyelectrolyte Nanoparticles Mediate Vascular Gene Delivery

AbstractPurpose. The purpose is to develop a non-viral gene delivery system that meets the requirements of colloidal stability of DNA complexes expressed in terms of no particle aggregation under physiologic conditions. The system should be used to transfect cardiovascular tissues. Methods. We used a strategy based on the formation of polyelectrolyte nanoparticles by deposition of alternatively charged polyelectrolytes onto a DNA core. Polyelectrolytes were transfer RNA as well as the synthetic polyanion, polyvinyl sulfate (PVS), and the polycation polyethylenimine (PEI). The PEI/DNA complex formed the DNA core. Results. We observed that the DNA is condensed by polycations and further packaged by association with a polyanion. These nanoparticles exhibited negative surface charge and low aggregation tendency. In vivo rat carotid artery experiments revealed high transfection efficiency, not only with the reporter gene but also with the gene encoding human urokinase plasminogen activator (Hu-uPA). Hu-uPA is one of the proteins involved in the recovery of the blood vessels after balloon catheter injury and therefore clinically relevant. Conclusions. A strategy for in vivo gene transfer is proposed that uses the incorporation of polyanions as RNA or PVS into PEI/DNA complexes in order to overcome colloidal instability and to generate a negative surface charge. The particles proved to be transfectionally active in vascular gene transfer.

Integrin specificity of the cyclic Arg-Gly-Asp motif and its role in integrin-targeted gene transfer.

Targeted gene transfer, addressing the αvβ3 integrin by coupling the appropriate ligand, cRGD (S2‐bridged cyclic Arg‐Gly‐Asp containing peptide) motif, on to a DNA condensing sequence was described as early as 1995 by Hart, Harbottle, Cooper, Miller, Williamson and Coutelle [(1995) Gene Ther. 2, 552–554]. Their work was followed by a series of publications, introducing the cRGD motif in polycationic DNA carriers, such as peptides, proteins and liposomes. Polyethylenimine and even adenoviruses were additionally ligated using the cRGD motif. ‘Integrin specificity’ has been determined from the significantly improved transfection efficiency compared with the DNA carriers with control ligands, mainly the cRGE (S2‐bridged cyclic‐Arg‐Gly‐Glu‐containing peptide) motif. However, by observing the physicochemical appearance of the resulting complexes and their controls such as the poly(l‐lysine)–DNA complexes carrying the cRGD and the cRGE motifs, we doubted the integrin‐mediated specificity of the increased transfection efficiency. To clarify this contradiction, we investigated the suitability of the cRGD motif for targeted gene transfer. We proved the specificity of the RGD motif and its controls using computational docking procedures and molecular modelling methods. Since we were confident of the motifs used, we improved our transfection method. Since aggregation of the RGD‐ligated poly(l‐lysine)–DNA complexes under physiological conditions caused an enormous amount of unspecific cell uptake and transfection, a method had to be designed to exclude aggregation processes of the motif‐polycation–DNA complexes. Small complex sizes are necessary for receptor‐specific uptake. The complexes were therefore recharged using poly(vinyl sulphate). Inhibited aggregation of the targeted DNA carriers under physiological conditions is a necessary prerequisite for successful in vivo gene transfer.

Ultrastructural analysis of DNA complexes during transfection and intracellular transport.

We present a simple method based on transmission electron microscopy that allows investigation of the early steps of polyplex-mediated transfection without the use of labeled DNA. The ultrastructural analysis showed internalization of 0.2–1-μm aggregates composed of 30–50-nm subunits. In addition, new details of the internalization process were revealed, suggesting an unspecific cell entry mechanism of large DNA aggregates.

Structural aspects of histone H1–DNA complexes and their relation to transfection efficiency

During transfection, polycation–DNA complexes are normally diluted by the transfection medium, which often contains salt in the physiological concentration range and serum. It is not exactly known to what extent this dilution step influences the properties of the complexes, which in turn influence the transfection efficiency. In order to gain more insight into the size–structure–transfection activity relationship, we prepared histone H1–DNA complexes in NaCl solutions at various concentrations known to determine the size and structure of the resulting complexes. We characterized the complexes by physicochemical methods. Fluorescence correlation spectroscopy enabled relative measurements of complex sizes even under physiological conditions. The different appearances of the complexes were correlated with their transfection efficiency. When transfection was performed by dilution of the complexes in cell‐cultivation media, the initial structure of H1–DNA complexes preformed under distinct salt conditions had no significant influence on the transfection efficiency. The dilution of the preformed complexes with cell‐cultivation medium resulted in re‐formation and aggregation of the complexes. The addition of the complexes to the cells without cell‐cultivation medium, however, showed a direct correlation between the size of the complexes and the transfection efficiency (corrrelation coefficient 0.91). Small complexes did not contribute to the transfection.

Brief Communication: Polycation-Mediated Transfection: How to Overcome Undesirable Side Effects of Sticky DNA Complexes

Using polycationic transfection one encounters undesired persistent binding to cells of sticky polycation/DNA complexes. These complexes simulate transfection under conditions where no uptake is expected e.g. at 4°C if the uptake is by endocytosis. To overcome this problem, using H1/DNA complexes, we developed an easy and nontoxic method for removing the sticky complexes not taken up during the transfection phase. The cells are simply washed with isotonic (0.1 M) MgCl2 solution, which enables the complete removal of the complexes by their rapid dissolution.