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354 Cell-Penetrating Peptides: Processes and Applications
Recently, Eguchi et al. 49 demonstrated utilization of Tat peptide for gene transfer
to various mammalian cell lines (Table 16.1). They constructed recombinant lambda
phage particles displaying Tat peptide on their surfaces and carrying mammalian
marker genes (GFP and firefly luciferase) as part of their genomes. In animal cells
briefly exposed to Tat–phage, the expression of phage marker genes was induced.
In contrast, recombinant phage displaying other functional peptides, like the peptide
from integrin-binding domain (RGD) or a nuclear localization signal (NLS) similar
to controls (wild type phage and naked DNA), could not induce detectable marker
gene expression. The expression of marker genes induced by Tat–phage is not
affected by temperature or by endosomotropic agents such as monensin and chloroquine
(for further reading, see Galloway et al. 50 ). However, expression is partially
impaired by inhibitors of caveolae formation such as nystatin 51 and fillipin. 52
The ability of Tat–phage to transport dsDNA was also shown in vivo. Constructs
carrying GFP gene were injected intraparenchymally into the mouse liver. Livers
were dissected in 2 days and the GFP expression observed by fluorescence microscopy.
These data suggest that Tat peptide could be used in gene transfer in vivo.
Also, the existence of classical endocytic pathway-independent mechanism for Tatmediated
uptake was obviously proved again.
16.4.3 TRANSPORTAN
Transportan is a 27-amino acid artificial chimeric peptide developed by the authors. 37
This peptide has been demonstrated to locate through biological membranes and finally
localize in the nucleus. The ability to transport antisense PNA molecules in vitro and
in vivo was demonstrated in the article published shortly after introducing the peptide. 44
This report compares penetratin and transportan as transmembrane delivery vectors for
PNA molecules, demonstrating similar effects with both vectors (see above).
A recent publication by Beletskii et al. 53 reported use of transportan to target
several PNA oligomers to diploid female ES cells (Table 16.1). The study demonstrates
the effect of PNA oligomers on inactivation of X chromosome (Xi) and
formation of macrochromatin body (MCB). This complex is formed to silence the
X chromosome in female mammals. Transportan coupled with 19-mer PNA complementary
to noncoding Xist RNA, a noncoding RNA responsible for Xi silencing,
inhibits Xist complexing with Xi. Such inhibition leads to disruption of association
between Xi and H2A macrohistone. These results prove the potential of transportan
as a cell-penetrating delivery vehicle.
16.4.4 MTS–NLS
Certain peptides from protein signal sequences have been shown to penetrate membranes.
Membrane translocating sequences (MTS) are used by protein sorting
machinery to target proteins into different intracellular compartments. Nuclear proteins
are usually tagged with nuclear localization signals (NLSs). MTS–NLS conjugates
have shown the ability to enter cells and localize in the nucleus. 54
Peptides containing a hydrophobic N-terminal part (signal peptide sequence of
Cayman crocodylus Ig(v) light chain) associated with an NLS (the C-terminal