crc press - E-Lib FK UWKS

348 Cell-Penetrating Peptides: Processes and Applications
proteins, thus preventing them from associating to their normal target. Oligonucleotides
can also recognize double-helical DNA at specific sequences by forming Hoogsteen or
reverse-Hoogsteen hydrogen bonds with purine bases on one of the duplex strands; this
forms a local triple helix, a strategy known as “antigene” approach.
Transfection of cis-element ODNs (decoy ODNs) has been recently accepted as
a powerful tool that could be useful in a new class of antigene strategies for gene
therapy and in the study of transcriptional regulation. These ODN decoys will disturb
the authentic cis–trans interaction leading to the removal of trans-factors from
endogenous cis-elements with subsequent modulation of gene expression. Morishita
et al. first demonstrated the use of decoy ODNs for the therapeutic manipulation of
gene expression in 1995. 2 They demonstrated treatment of rat arteries with ODNs
bearing the consensus of binding site for the E2F family of transcription factors.
Recently several groups have reported on decoy ODN as an in vivo gene therapy
reagent further highlighting therapeutic potential of these oligonucleotides. 3-7
Targeting oligonucleotides to the DNA or DNA-binding molecule (the antigene
or decoy) has advantages compared to targeting antisense ODNs:
1. There are only two alleles of the targeted gene, whereas there may be
thousands of copies of an mRNA. Blocking mRNA translation by inducing
sequence-targeted cleavage of the RNA chain does not stop the respective
gene from transcription.
2. Gene transcription regulation is thought to bring the mRNA concentration
down more efficiently and the effect is believed to last longer.
However, the therapeutic potential of ONs will not be fulfilled until their uptake
by cells has been considerably improved. Recently, CPPs have been applied to
improve the ON uptake.
16.2 TRANSPORT OF NAKED OLIGONUCLEOTIDES
Cellular delivery of naked ONs has been thought to be too slow for use in therapeutic
applications. ONs are known to be transported through the lipid bilayer where the
major mechanism behind an uptake of negatively charged ONs is receptor-mediated
endocytosis. 8,9 Unfortunately, this process is energy-dependent and saturable.
Adsorptive endocytosis and pinocytosis also contribute to ON uptake in a process
involving a membrane protein. It has been demonstrated that a 30-kDa DNA-binding
membrane protein 10,11 enhances the uptake of DNA molecules longer than 7 kb; this
effect could be inhibited by other negatively charged molecules. 12
Finally, reports exist that demonstrate different mechanisms for ON uptake not
related to receptors. Some of these show internalization of ONs by the proteinindependent
mechanism of endocytosis or internalization by a caveolar, potocytotic
mechanism rather than by receptor-mediated uptake. 13 Others have demonstrated the
involvement of different transporter proteins 14-16 or pore-forming proteins. 17,18 Such
contradiction in published results could be a consequence of cell-specific involvement
of different ON transport pathways or different experimental conditions.