IMPROVING CELLULAR UPTAKE
Beside metabolic elimination by nucleases, the poor cellular uptake of ONs poses a problem for therapeu-tic application of ONs. Compared to conventional drugs, ONs are relatively large and polyanionic, making passage over cellular membranes virtually impossible. Cellular uptake of ONs can be improved in vitro and in vivo by physical methods, chemically modifying the ONs or by making use of specialized delivery systems.
Electroporation of tissue after local injection of ONs results in improved cellular uptake. Due to high voltage pulses, transient perforations in the cell membrane occur that allow passage of ONs into the cytosol. This technique can of course only be appliedfor delivery of ONs in vitro and in vivo to tissues readily available for electroporation (e.g., skin, skele-tal muscle, or superficial tumor tissue).
Grafting ONs with cationic groups in order to reduce the ionic repulsion between ON and cell membrane represents an alternative strategy to enhance cellular uptake. Synthetic guanidinium-containing ONs (GONs) showed improved duplex and triplex stability in addition to enhanced cellular uptake (Deglane et al., 2006). The uptake pattern suggests that these cationic ONs are internalized by endocytosis, although cytosolic localization could also be observed.
Alternatively, cell-penetrating peptides (CPPs) can be conjugated to ONs with the purpose to enhance membrane translocation. CPPs are small basic pep-tides derived from protein transduction domains present in a variety of proteins which have strong membrane translocating properties. Conjugation of such a CPP called transportan to PNAs resulted in enhanced cellular uptake in a dose-dependent manner with preservation of antisense activity of the PNA.
Lipid modification of siRNA has also been proven to be beneficial for cellular uptake and subsequent gene silencing. siRNA against apoB mRNA, which was modified by attaching a cholesterol group to the 30 terminus of the sense strand, showed increased silencing of the gene encoding for apolipoprotein B compared to the unmodified siRNA after intravenous injection into mice (Soutschek et al., 2004).
Another strategy involves the use of sophisticated delivery systems to enhance cellular uptake and to target ONs to specific tissues or cells. Most of the delivery systems for ONs are based on complexation of ONs with cationic molecules, of which cationic lipids (e.g., LipofectamineTM) are the most common. The complexation has a dual function: it protects the ONs from nuclease attack and enhances cellular internaliza-tion. By shielding the cationic complexes with large polyethylene glycol polymers displaying targeting ligands, such complexes can be targeted to specific cell types. This has been demonstrated for the delivery of siRNA to angiogenic vascular endothelial cells (Schiffelers et al., 2004). Targeting can also be accom-plished by covalently coupling the ONs to antibodies. In this way, PNAs conjugated to an anti-transferrin receptor monoclonal antibody could be transported over the blood–brain barrier (Shi et al., 2000).
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