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4 Cell-Penetrating Peptides: Processes and Applications
(IFN) inducers, 2-5A activators of RNaseL, and antisense oligonucleotides. In this
context, an early experiment indicates how appropriate delivery could benefit the
field of nucleic acids therapeutics. Synthetic double-stranded RNA (ds RNA) as
poly(I).poly(C) have been used as IFN inducers in several cell cultures or even in
animal models. Approximately 10 5 molecules of poly(I).poly(C) are requested to
induce IFN production 2 when incubated in a cell culture model, while a few molecules
suffice to promote IFN induction when poly(I).poly(C) is directly microinjected
into HeLa cell nuclei. 3 Somatic cell microinjection cannot be applied to a large
number of cells and is therefore limited in its application; hence the search for
alternative routes to bypass cellular membranes.
Various polycationic polymers have proven useful to promote the internalization
of drugs or even macromolecules. One of the most extensively studied in this respect
has been poly(L-lysine) following initial studies by Shen and Ryser. They established
that chemical conjugation of methotrexate or proteins to poly(L-lysine) allowed
significantly improved internalization in various cell types. 4,5
We adapted this strategy to the intracellular delivery of 2-5A oligonucleotides
(ODNs) 6 and antisense ODNs. 7 A large improvement in cytoplasmic delivery and
biological activity was recorded in these experiments. Uptake took place through
adsorptive endocytosis; release of the conjugated oligonucleotide probably took
place in an endocytic compartment through degradation of most (if not all) of the
polybasic amino acid carrier by cellular esterases. Poly(L-lysine)-based delivery
vectors have also been engineered for the delivery of plasmid DNA, but transfection
yields were low compared to those of viral vectors. 8
Moreover, internalization through an endocytic compartment leads to entrapment
and, to a large part, degradation of the transported biomolecules in lysosomes.
Combination with endosome-disrupting agents or amphipathic fusogenic peptides
improved transfection efficiency at the expense of additional complexity of these
synthetic delivery vehicles (reviewed in Curiel 9 ). Despite numerous studies, the
future of this strategy for the systemic delivery of genes or other drugs is hampered
by serious drawbacks such as complexity, toxicity for certain cell lines, and immunogenicity
including complement activation.
A new strategy has emerged in recent years starting from the serendipitous and
surprising observation that some cellular proteins could be taken up by intact cells
when incubated in the culture medium. Interestingly, two of the most studied cellpenetrating
proteins are trans-activating factors: the Drosophila Antennapedia protein
(whose properties will be reviewed extensively here) and the human immunodeficiency
virus (HIV)-coded Tat regulatory protein.
1.2 TAT CELLULAR UPTAKE: FROM INITIAL DATA
ON THE FULL-LENGTH TAT PROTEIN TO THE DEFINITION
OF A 9-MER CELL-PENETRATING PEPTIDE
The HIV-coded Tat regulatory protein has been extensively studied by virologists
since the deletion of its gene has shown that it was essential for virus replication. 10
It is an 86-amino-acids-long nuclear protein which binds to the viral TAR region,