Proton Spongemediated Vacuole Escape

After internalization, DNA complexes must escape from the formed intracellular vacuoles (Fig. 1). Many viruses that do not fuse with the plasma membrane exploit endosome acidification as a signal triggering activation of an escape mechanism. This requires sophisticated conformational changes of fusion proteins. In the case of cationic lipids or polymer-DNA complexes, most particles probably remain trapped within vacuoles. DNA being protected within the complexes, this may even be regarded as a ''slow release'' process that sustains gene expression. In effect, cell division, although favoring initial transfection (see below), also leads to loss of the transgene(s) present in the nucleus of a transfected cell. During mitosis of a transfected cell, the intracellular events of gene delivery must thus occur again for that cell to remain transfected, hence the potential importance of slow release. Cationic lipids may possess some intrinsic bilayer-disrupting property, especially when forming nonlamellar phases [e.g., lipopolyamines form direct hexagonal phases (11), DOPE forms an inverted one (33)]. Subsequent vacuole rupture al lows charge neutralization of the cationic lipid by intermixing with anionic phosphatidylserine from the vacuole outer leaflet. This may liberate in part the complexed polynucleotide (34).

Cationic polymers possess no fusogenic activity per se. This is why fully cationized polymers such as polylysine require chloroquine, a lysosomotropic drug used to unmask the intravacuolar malaria parasite, to become effective transfec-tion agents. However, some cationic polymers (35), such as polyamine dendrimers (36,37) or PEI(38), share with chlo-roquine the ability to buffer the acidity of endosomes. Vacuolar pH decrease (39) should therefore coincide with a large ionic concentration increase. Osmotic swelling due to water entry may then burst some of the vacuoles and release the complex into the cytoplasm. This ''proton sponge'' hypothesis (38) proved to be fruitful, as it led to the design of other efficient polymeric vectors (40-42). It was recently supported by the fact that PEI transfection efficiency is 100-fold decreased by bafilomycin A, a specific vacuolar H + -ATPase inhibitor (43,44).

Over the years, PEI, and especially linear PEI (45-47) has become a versatile vector for in vitro and in vivo gene delivery, with over 300 publications referring to the use of its gene delivery properties. As a leading member of what can be regarded as the second generation of nonviral vectors for gene therapy, in vivo data using PEI deserve a paragraph.

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