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Signal Sequence-Based Cell-Penetrating Peptides 103
1- DNA
d i
lytic
condensing peptid
ligand
2- Cell targeting
DNA
3-Endosomolysis
4-Nuclear translocation
FIGURE 5.2 The four major steps in peptide-based gene delivery: (1) DNA binding and
condensation, (2) cell targeting, (3) endosomal release of the plasmid or ODN, and (4) nuclear
targeting.
cationic moiety, such as the polylysine DNA-binding domain. Noncovalent attachment
of an NLS to DNA is preferable to enable complete release of DNA from the
NLS-containing peptide following nucleocytoplasmic transport. However, a series
of parameters need to be controlled and cannot be generalized, including the number
of NLS molecules used and the ratio of NLS to DNA. Special attention is required
when considering the mechanism of complex formation and the order of association
of a multicomponent DNA carrier. The total amount of NLS is important because
the import mechanism can be saturated by an excess of free NLS. Also, care has to
be taken to avoid binding of the NLS within the DNA.
The NLS from the large T antigen of SV 40 (PKKKRKV) is one the most used
in nonviral gene delivery systems. 4,5,9,81 Peptides containing NLS are able to condense
DNA and to form a single transfection system without any further requirement. They
have been shown to increase aggregation at the surface of the cell and to improve
delivery. However, among the several examples described so far, the role of the NLS
in nuclear accumulation of DNA appears to be controversial. 82,83 The association of
an NLS to DNA was shown to increase transgene expression following cytoplasmic
injection into zebrafish eggs. 84 Complexes of DNA with peptide scaffolds consisting
of the M9 import sequence with a scrambled SV40 NLS peptide were used to
improve binding via NLSs. 9,85 Peptides or proteins containing NLS, complexed or
not to lipids, are used to improve nuclear transport through electrostatic binding of
plasmids. 9 An NLS associated to a PNA sequence with a sequence-specific DNAbinding
domain allows control of the number of attached molecules and of their
binding sites in the DNA. 66,67