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102 Cell-Penetrating Peptides: Processes and Applications
A similar strategy has been used to develop a short amphipathic peptide carrier for
protein delivery. This peptide carrier (chariot) contains three domains: a hydrophobic
tryptophan-rich motif, an NLS domain derived from the SV40 large T antigen, and
a spacer domain which improves flexibility and integrity of the hydrophobic and the
hydrophilic domains. Chariot is able to efficiently deliver a variety of peptides and
proteins into several cell lines in a fully biologically active form, without the need
for prior chemical covalent coupling or denaturation steps. 52
5.4 UTILIZATION OF THE NLS PATHWAY FOR NUCLEAR IMPORT
OF GENE DELIVERY VECTORS
5.4.1 CHALLENGES IN GENE DELIVERY
In the past 5 years the potential hazards of viral vectors as well as their immunogenicity
have led research in the field of gene therapy to focus at least in part on
the design of new tools and methods for gene delivery. In this respect, the major
challenge resides in the design of nonviral vectors that can compensate for the poor
permeability of the cell membrane to nucleic acids and improve intracellular trafficking
and nuclear delivery of genes into target cells with minimal toxicity. 5,9,76-78
Nonviral systems present several advantages over viral systems, in that they are
simple to use, easily produced, do not induce specific immune responses, and are
less cytotoxic. Cell-penetrating peptides constitute an interesting subclass among
nonviral gene delivery systems. The challenge in peptide-based gene delivery systems
is to define the ideal formulation that satisfies most of the major requirements:
(1) reversible binding to cargo or DNA, (2) cellular or tissue specificity essential
for in vivo gene delivery, (3) membrane fusogenic or disruptive activities, and (4)
ability to promote nuclear translocation of plasmids (Figure 5.2).
One of the major issues in gene therapy is the dependency of delivery systems
on the mitotic activity of cells, illustrated by lack of efficient delivery into nondividing
cells. This is primarily due to the inability of most nonviral gene delivery
systems to translocate plasmids into the nucleus of nondividing cells. Several modifications
of plasmid DNA have been proposed to improve their interaction with the
nuclear import machinery, including additional cloning of specific DNA sequences
recognized by transcription factors, the attachment of glycosylated moieties, or of
synthetic peptides containing a nuclear localization signal. 79,80 Alternatively, NLSs
have been incorporated into nonviral gene delivery systems and either noncovalently
or covalently linked to the cargo or the DNA. Except for a few examples, incorporation
of an NLS significantly increases the efficiency of transfection. However, in
contrast to their effect on proteins, NLSs do not appear to improve nuclear targeting
of DNA; significantly in most cases, NLS simply improves formation of DNA and
peptide complexes through electrostatic interactions. 8,9
5.4.2 NONCOVALENT INCORPORATION OF NUCLEAR LOCALIZATION SEQUENCES
Noncovalent attachment of the NLS can be achieved by taking advantage of the
cationic residues within the NLS or by fusion or covalent binding of the NLS to a