Endosomes, lysosomes: their implication in gene transfer
Introduction
During transfection, exogenous plasmid DNA is probably taken up either by endocytosis or phagocytosis except when aggressive methods like electroporation, impossible to apply in vivo, are used [1], [2], [3]. This is true for both naked nucleic acid molecules or those associated with a vector. Plasmid DNA, like other endocytosed molecules, travels after internalisation in the cell through an endosomal compartment before reaching lysosomes where it is degraded (Fig. 1). On the other hand, for a transfected gene to be expressed, a prerequisite is that some intact DNA molecules should cross either the endosomal or lysosomal membrane into the cytosol and later find itself in the nucleus. Therefore, factors affecting endosomal and lysosomal systems could either facilitate release of plasmid DNA into the cytosol or protect these molecules from degradation by lysosomal nucleases thereby influencing the efficiency of transfection. In this paper, we will consider some of these factors showing how they could affect transfection.
Section snippets
Effects of non-viral vectors
Frequently, plasmid DNA used for transfection is combined with a carrier: cationic lipids, polycations [4], [5], [6]. The complex makes the DNA compact, it bears a positive net charge and prevents to some extent the hydrolysis of DNA by nucleases. On the other hand, efficiency of transfection is probably dependent on the rate of intracellular degradation of plasmid DNA. How the type of cationic compound influences the intracellular degradation of plasmid DNA is poorly documented. For example,
Destabilisation of endosomal and lysosomal membranes
When plasmid DNA is enclosed in endosomes or lysosomes its escape into the cytosol will be made easier if membrane-destabilising compounds are simultaneously present in the organelles. It is well known that some viruses and peptide toxins use a fusogen peptide to cross endosomal membrane and reach the cytosol. This is the reason why some transfection methods take advantage of the endosomolytic power of replication defective viruses [13] or make use of fusogenic peptides derived from virus [14],
Lysosomotropic compounds
Lysosomotropic substances are inclined to accumulate in lysosomes [21]. There are two kinds of such compounds differing by their mode of entry into the cell and the lysosomes. The first category concerns molecules that reach lysosomes by the endocytic pathway, the second type of compounds cross the plasma membrane and the lysosomal membrane by diffusion.
Most endocytosed molecules are digested by lysosomal hydrolases and the hydrolysis products diffuse into the cytosol. Nevertheless, certain
Nucleases
Until now, among the subcellular structures involved in the traffic of endocytosed compounds, only lysosomes have been described as containing nuclease: DNAse II, exonuclease. If this is true, plasmid DNA taken up by endocytosis will be solely hydrolysed in these organelles, at least before reaching the cytosol. Substances able to inhibit plasmid DNA degradation by lysosomal nucleases should increase transfection efficiency. Recent results of Ross et al. [30] suggest the possible use of acid
Cellular and intracellular targeting
Endocytosis can be achieved by a fluid phase, an adsorptive, or a receptor mediated process. Fluid phase endocytosis concerns any non-diffusable molecule present in the extracellular medium. Adsorptive endocytosis involves the nonspecific binding to plasma membrane of the compound that has to be taken up. Receptor-mediated endocytosis requires the presence in the plasma membrane of specific receptors that link the molecule destined to be endocytosed. Obviously, adsorptive- and receptor-mediated
Conclusions
When transfection is achieved with viral vectors, the viral machinery allows DNA to have access to the cytosol. Obviously, the problem is different when naked plasmid DNA or plasmid DNA complexed to non-viral vectors is used. Indeed, as DNA enters the cell by endocytosis, something must happen during the intracellular journey of the macromolecule that will lead to its escape into the cytosol preferentially before they reach lysosomes. In our opinion, until now there are no clearcut explanations
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