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crc press - E-Lib FK UWKS

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348 Cell-Penetrating Peptides: Processes and Applications<br />

proteins, thus preventing them from associating to their normal target. Oligonucleotides<br />

can also recognize double-helical DNA at specific sequences by forming Hoogsteen or<br />

reverse-Hoogsteen hydrogen bonds with purine bases on one of the duplex strands; this<br />

forms a local triple helix, a strategy known as “antigene” approach.<br />

Transfection of cis-element ODNs (decoy ODNs) has been recently accepted as<br />

a powerful tool that could be useful in a new class of antigene strategies for gene<br />

therapy and in the study of transcriptional regulation. These ODN decoys will disturb<br />

the authentic cis–trans interaction leading to the removal of trans-factors from<br />

endogenous cis-elements with subsequent modulation of gene ex<strong>press</strong>ion. Morishita<br />

et al. first demonstrated the use of decoy ODNs for the therapeutic manipulation of<br />

gene ex<strong>press</strong>ion in 1995. 2 They demonstrated treatment of rat arteries with ODNs<br />

bearing the consensus of binding site for the E2F family of transcription factors.<br />

Recently several groups have reported on decoy ODN as an in vivo gene therapy<br />

reagent further highlighting therapeutic potential of these oligonucleotides. 3-7<br />

Targeting oligonucleotides to the DNA or DNA-binding molecule (the antigene<br />

or decoy) has advantages compared to targeting antisense ODNs:<br />

1. There are only two alleles of the targeted gene, whereas there may be<br />

thousands of copies of an mRNA. Blocking mRNA translation by inducing<br />

sequence-targeted cleavage of the RNA chain does not stop the respective<br />

gene from transcription.<br />

2. Gene transcription regulation is thought to bring the mRNA concentration<br />

down more efficiently and the effect is believed to last longer.<br />

However, the therapeutic potential of ONs will not be fulfilled until their uptake<br />

by cells has been considerably improved. Recently, CPPs have been applied to<br />

improve the ON uptake.<br />

16.2 TRANSPORT OF NAKED OLIGONUCLEOTIDES<br />

Cellular delivery of naked ONs has been thought to be too slow for use in therapeutic<br />

applications. ONs are known to be transported through the lipid bilayer where the<br />

major mechanism behind an uptake of negatively charged ONs is receptor-mediated<br />

endocytosis. 8,9 Unfortunately, this process is energy-dependent and saturable.<br />

Adsorptive endocytosis and pinocytosis also contribute to ON uptake in a process<br />

involving a membrane protein. It has been demonstrated that a 30-kDa DNA-binding<br />

membrane protein 10,11 enhances the uptake of DNA molecules longer than 7 kb; this<br />

effect could be inhibited by other negatively charged molecules. 12<br />

Finally, reports exist that demonstrate different mechanisms for ON uptake not<br />

related to receptors. Some of these show internalization of ONs by the proteinindependent<br />

mechanism of endocytosis or internalization by a caveolar, potocytotic<br />

mechanism rather than by receptor-mediated uptake. 13 Others have demonstrated the<br />

involvement of different transporter proteins 14-16 or pore-forming proteins. 17,18 Such<br />

contradiction in published results could be a consequence of cell-specific involvement<br />

of different ON transport pathways or different experimental conditions.

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