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392 Cell-Penetrating Peptides: Processes and Applications
(PNA) in E. coli. Antisense agents are designed to inhibit gene expression through
sequence-specific nucleic acid binding, which normally requires formation of ten or
more base pairs. Oligonucleotides of such lengths are typically too large for efficient
passive cellular uptake by diffusion across lipid bilayers; cellular outer membrane
structures can pose additional barriers. 67
Recently, we introduced peptide nucleic acids (PNA) as antisense agents for
bacterial applications. 68 Early experiments indicated very encouraging sequence
specificity against reporter genes; however, uptake was limited by bacterial cell
barriers. 69 This limitation is not surprising as E. coli and other Gram-negative bacteria
have outer and inner bilayer membranes; the outer membrane contains a lipopolysacharide
layer that stringently restricts the entry of foreign molecules. 12
To provide carrier domains to improve cell uptake and potency, we first selected
a range of short, cationic peptides that appeared to have cell-permeabilizing or cellpermeating
activities against E. coli. These peptides were attached to PNA via
flexible linkers. 6 Several of the peptides selected, in particular the KFFKFFKFFK
peptide, provided efficient carriers for PNA. Indeed, an antisense peptide–PNA
targeted to the essential acpP gene is bactericidal and shows encouraging bacterial
cell specificity. 70,71 E. coli cells were grown in mixed culture with HeLa cells and
the peptide–PNA efficiently cleared the human cell culture of inoculated bacteria
without apparent harm to the HeLa cells, even when present at a concentration that
was ten-fold higher than needed to kill the bacteria. 6 Therefore, microbial cellpermeating
peptides can act as carriers for antisense agent delivery and can display
toxic effects that appear largely specific against microbial cells.
ACKNOWLEDGMENTS
We thank the Swedish Science Council, Swedish Foundation for Strategic Research,
and Pharmacia Corporation for support and our collegues for helpful comments on
the manuscript.
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