Histone H1-mediated transfection: serum inhibition can be overcome by Ca2+ ions.

PURPOSE: One of the drawbacks of polycationic and cationic liposomal gene transfer is its sensitivity to serum. Gene therapy requires the transfectant-DNA complex to be resistant to serum as well as blood. Since Ca2+ has proved to be an efficient cofactor of polycationic gene transfer, we decided to investigate its effects on transfection in the presence of serum. METHODS: We studied transgene expression of luciferase gene (pCMV Luc) on ECV 304 human endothelial cells using H1 histone and DOSPER as transfectants in the presence of 0-100% fetal calf serum. RESULTS: H1-and DOSPER-mediated transfection was found to be inhibited by serum above the concentration of 10%. If 2 mM Ca2+ or 2 mM Ca2+/0.1 mM chloroquine was included in the culture medium which replace the transfection mixture and was left on the cells for 24 hours postincubation, the inhibiting effect of even 100% serum was overcome. CONCLUSIONS: A high serum level does not interfere with binding and uptake of H1- and DOSPER-DNA complexes, but inhibits subsequent steps such as endosomal escape. Ca2+ in the form of nascent calcium phosphate microprecipitates and other lysosomolytical agents facilitate endosomal/lysosomal release by their fusigenic and membranolytic activity.

Calcium ions as efficient cofactor of polycation-mediated gene transfer.

We investigated the effect of calcium on the transfection of non-viral DNA transfer systems. Cationic proteins such as the nuclear protein H1, the polycation polylysine and a number of commercial transfection agents exhibited high transfection rates in the presence of Ca2+. Without Ca2+ H1 and HMG1 were inactive in transfection of the human permanent endothelial cell line ECV 304 while cationic liposomes such as Lipofectin and Lipofectamine did not show any Ca2+ dependence. More detailed experiments showed that Ca2+ was replaceable by the lysosomotropic agent chloroquine. Furthermore, it was possible to separate the transfection-enhancing role of Ca2+ from the actual transfection process by adding Ca2+ to the cells after the transfection period and still to obtain a significant transgene expression. This makes it possible to distinguish between cellular uptake of H1 (or mediator)-DNA complexes and endocytotic release. We also replaced soluble Ca2+ by Ca-phosphate precipitates not containing DNA and obtained similar transfection results. This allowed us to suggest that the addition of free Ca2+ to the transfection medium resulted in nascent Ca-phosphate microprecipitates. The known fusogenic and membranolytic activity of such microprecipitates could facilitate the transport through and the release of the transfecting complexes from the endosomal/lysosomal compartment.

The encapsidation signal on the hepatitis B virus RNA pregenome forms a stem-loop structure that is critical for its function.

Hepatitis B virus (HBV) is the type member of the hepadnaviridae, small enveloped DNA viruses that replicate through reverse transcription of an RNA intermediate, the pregenome. This reaction occurs usually inside the viral nucleocapsid, the assembly of which requires specific interactions between multiple copies of the core protein, the viral replication enzyme (P protein) and the RNA pregenome which also serves as mRNA for both proteins. Deletion studies have established that specific packaging of the RNA is mediated by a short cis-acting sequence, the encapsidation signal epsilon. Using nuclease sensitivity experiments we provide experimental evidence that part of this sequence can adopt a stem-loop structure that is interrupted by a bulge and a single unpaired U residue. The structural consequences of deletions of the unpaired regions and changes in their primary sequences were investigated in vitro, and their influence on the function of the epsilon-signal was tested in animal cells by monitoring encapsidation of RNAs carrying the mutant epsilon-sequences in front of a 2.7 kb foreign RNA fragment, or within the context of a complete HBV genome. The data indicate that the entire stem-loop structure containing the bulge and the loop is critical for encapsidation competence. While gross alterations in the primary sequences of the unpaired regions interfere with encapsidation, data obtained with additional mutants suggest that the bulge region is more tolerant to sequence changes than the loop.