Quantitative studies on the nuclear transport of plasmid DNA and gene expression employing nonviral vectors
Introduction
Cationic liposomes, an attractive non-viral vector, have several advantages, which include low immunogenicity, ease of handling and the availability of large-scale preparations. However, the efficiency of transfection is lower than that of a viral vector. A number of cationic liposome/lipid formulations have been described [1], [2] and liposomes have been modified with ligands or monoclonal antibodies for receptor-mediated targeting [3], [4], [5], polyethyleneglycol for circumventing reticuloendothelial system (RES) uptake [6], [7], and materials such as hemagglutinin or a GALA peptide for enhancing the endosomal escape of cationic liposomes/plasmid DNA complexes [8], [9], [10] in order to increase the efficiency of gene expression. However, the level of transfection efficiency is currently not sufficient high for actual gene therapy. Since non-modified liposomal vectors, unlike viral vectors, per se do not have any devices for regulating intracellular gene trafficking, it is necessary to optimize the liposomal vector for their regulation.
Generally, higher levels of reporter gene expression are observed in actively dividing cells such as cancer cells than nondividing cells in the case of lipofection. It has been proposed that the nuclear uptake of exogenous DNA occurs only in cells that are actively replicating and that the nuclear envelope disappears during mitosis [11], [12], [13]. In fact, however, the exact mechanism of the nuclear delivery of DNA into the nucleus is not clear, since some publications suggest that cell mitosis is not an absolute requirement for gene expression [14], [15].
To achieve gene therapy on a widely applicable scale, increasing the transfection efficiency in nondividing cells is essential because a large fraction of the target cells of the organism are in that state. For this purpose, the transport of exogeneous DNA to the nucleus via the nuclear membrane is an important issue. Recent studies suggest that the nuclear membrane is a serious barrier for exogenous gene expression [15], [16], [17], and attempts, especially those which employ the nuclear localization signal (NLS), have been made to overcome this barrier.
To more clearly understand the mechanism of the nuclear transport of exogenous DNA and to improve the gene expression efficiency in nondividing cells, the quantitative evaluation of intracellular trafficking of introduced plasmid DNA, particularly the relationship between the entry of the plasmid DNA into the nucleus and the resulting gene expression, is crucial. However, to date, only the final output, i.e., enzymatic activity of the resulting product of a reporter gene such as luciferase or chloramphenicol acetyltransferase has been measured, and the nature of the intracellular events remain highly speculative. Little is known about the process after the entry of plasmid DNA to the nucleus.
Here, we review recent work on the entry mechanism of plasmid DNA in the nondividing phase, the quantitative evaluation of the delivery of the plasmid DNA to the nucleus and the effect of the state of the plasmid DNA delivered to the nucleus on transcription.
Section snippets
The transport of exogenous DNA via nuclear pore complex
The nucleus contains a double membrane, the nuclear envelope. Nuclear pore complexes (NPCs), which have a diameter of 9 nm, are present in the nuclear envelope and are involved in the transport of substances. Molecules smaller than 40–45 kDa can apparently diffuse freely into and out of the nucleus. Karyophilic proteins larger than 45 kDa can be actively transported through the nuclear pores. The selective, active transport of nuclear protein is mediated by a nuclear localization signal (NLS)
Quantitative evaluation of the delivery of the plasmid DNA to the nucleus
Studies on the fundamental mechanisms of gene delivery are very important. A knowledge of each process in intracellular gene delivery will, most likely, lead to the development of nonviral vectors which can be successfully be used. In early reports, radio-labeled or fluorescence-labeled plasmids were used for the quantitative analysis of plasmids delivered into cells and only the amount of intracellular delivered plasmids, namely the uptake, was measured [3], [31]. However, it is difficult to
Effect of nonviral vectors on the processes after the entry of the plasmid DNA into the nucleus
In above sections, we discussed strategies to overcome the nuclear membrane, which is one of the major barriers in gene delivery using nonviral technology. Furthermore, it is very important that an understanding of the nature of the plasmid DNA delivered to the nucleus and the effects of chemical modification of plasmid DNA and/or cationic liposome delivered with plasmid DNA on the process after the nuclear transport of plasmid DNA, namely transcription. The issue of the form of the plasmid DNA
Conclusion
In this review, efficient gene delivery with nonviral vectors to nondividing cells has been discussed. It was concluded that (1) gene delivery using a nuclear localization signal, (2) quantitative evaluation of plasmid DNA delivered to the nucleus and the relationship between the amount of plasmid DNA delivered into the nucleus and gene expression, and (3) a method for the evaluation of the effect of the state of plasmid DNA on transcription in vitro are the important issues.
The nuclear
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