Cell Targeting And Entry

To enter their hosts, many viruses and bacteria bind to cell/matrix- or cell/cell-anchoring proteins, such as heparansulfate proteoglycans (HSPGs) and integrins. Anionic HSPGs are involved in cell entry of species with sizes ranging from chlamydia trachomatis (7) to adeno-associated virus (8). Interestingly, condensed DNA particles also require a cationic surface for gene delivery to occur (9), and their cell entry was shown to be mediated by electrostatic interaction with HSPGs

(10-12). This is presumably true for all polycationic entities, including calcium phosphate-precipitated DNA.

A common receptor suggests a single mechanism of cell entry. Among ubiquitous HSPGs that may act as receptors are syndecans. This family of transmembrane proteins can cluster to form focal adhesions following individual electrostatic binding to a large polycationic entity. There is evidence (13-15) that syndecans clustering induces their binding to the actin cytoskeleton and eventually the formation of tension fibers. Yet here tension would not provide cell anchoring,

Figure 2 DNA is condensed by thiol-containing oligocations presenting a low binding cooperativity. After equilibration, the particles are ''frozen'' by DNA template-assisted oxidation of thiols into disulfides. Transmission electron microscopy of the particles obtained from a 5.5-kbp plasmid shows a homogeneous population of 25-nm spheres.

Figure 2 DNA is condensed by thiol-containing oligocations presenting a low binding cooperativity. After equilibration, the particles are ''frozen'' by DNA template-assisted oxidation of thiols into disulfides. Transmission electron microscopy of the particles obtained from a 5.5-kbp plasmid shows a homogeneous population of 25-nm spheres.

but rather a mechanism and the energy to engulf the cationic particle and form an intracellular vacuole (Fig. 1). That cell anchoring and cationic particle binding are competitive processes involving HSPGs may explain why cells have the tendency to detach from their substratum during transfection.

Unfortunately, in vivo a cationic DNA-containing particle will quickly be bound to circulating or extracellular matrix polyanionic proteins. This prevents most particles from reaching their cellular target and eventually releases DNA from the complexes. Therefore, anionic receptors are generally too ubiquitous molecules to serve as targets for synthetic vectors. As mentioned above, integrins share many functional properties with syndecans. Several integrins bind and internalize zwitterionic RGD peptide-presenting particles in vivo (16). The mechanism may be identical to the one described above for HSPGs i.e., mediated by integrin ligation and actin fibers retraction). Imititating adenovirus, RGD peptides have been chemically conjugated to polycations such as polylysine (17,18) or polyethylenimine (PEI) (19,20) and complexed with DNA. The resulting particles were shown to deliver genes to epithelial cells in culture up to 100-fold better than the corresponding polycation-DNA complexes (Fig. 3). Control experiments using RGE-coated particles confirmed the enhanced transfection to be due to avps integrin-mediated cell entry.

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