Biomedical Engineering Reference
In-Depth Information
polylysine-Antp (
L
-Antp) in gene delivery, and comparable or superior to a
commercial liposome.
4.5.3 Nuclear Localization Signal
Nonviral gene therapy is challenged by inefficient delivery at the level of
intracellular processing. Several barriers have been described and studied,
including failure to escape from vesicular structures, lysosomal degradation,
enzymatic degradation in the cytosol, entrapment in the highly viscous and
crowded cytosol, lack of transport towards the nucleus and uptake into the
nucleus, and finally inefficiency of transcription and/or translation. Capecchi
et al. found that transport into the nucleus is the major bottleneck for
successful nonviral gene delivery.
96
The nuclear localization signal (NLS),
which is recognized by nuclear transport proteins (e.g., importin-a which
directly binds the NLS signal; and importin-b, which is the mediator of the
actual import process through the nuclear pores) could overcome the nuclear
membrane barrier and promote the nuclear translocation (Figure 4.9). Among
them, the extensively studied NLS sequence is the SV40 large-T antigen
(especially in its shortest form, Pro-Lys-Lys-Lys-Arg-Lys-Val).
Much effort has been made to mimic this process by incorporating NLSs in
gene delivery complexes. Dean et al. first demonstrated that plasmid nuclear
import could be facilitated by the insertion into the plasmid of nuclear DNA-
targeting sequences.
97
Recently, Wang and co-workers introduced an iodine
atom to a nuclear localization signal (PKKKRKV) by chemically attaching 2-
iodobenzoic acid to the peptide to obtain NLS-I peptide for cell targeting and
nuclear transport.
98
They found that cell internalization and nuclear
accumulation of NLS-I was markedly increased compared to NLS in MCF-
7 cells, and gene expression by PEI1800/DNA/NLS-I complexes exhibited
much
d
n
4
y
3
n
g
|
3
enhanced
efficiency
(up
to
130-fold).
This
study
demonstrates
an
Figure 4.9
Nuclear localization signal-mediated nuclear import.
