Challenges and Opportunities in Gene Delivery - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 68

triggers back the release of the imported polypeptides from importins, thus releasing
the free DNA for transcription [177] . Attachment of NLS to plasmid DNA and DNA
fragments can stimulate both the nuclear accumulation and expression of plasmid
DNA, consistent with the notion that DNA molecules traverse the NPC [36,178-180] .
After covalent joining of single or multiple classical NLS (SV-40 T antigen type) to the
DNA, enhanced transfection efficiency, presumably via the importin-dependent nuclear
transport pathway, was observed. However, after using the nonclassical NLS (the M9
sequence of the human heterogenous nuclear ribonucleoprotein A1), similar transfec-
tion efficiency was observed in the nondividing endothelial cells [181] . Although con-
densation, and thus protection of plasmid DNA by the positively charged linker peptide
comprising the NLS, may account in part for the effect, the majority of it could be
attributed to the activity of the NLS by transporting the DNA to the nucleus. Replacing
critical amino acid residues in the NLS abolished the nuclear targeting of the peptide
[36] , thus indicating a very unique transportation system specific for the targeted cells.
Although direct comparison of the efficacy of synthetic peptides and transcription fac-
tors is not feasible, these results suggest that combination of transportin-, importin-
and transcription factor-dependent nuclear targeting may have an additive effect on the
nuclear uptake capacity of the nonviral delivery system. The above experiments not
only verified that plasmid DNA can enter the nucleus by translocation via NPCs in
nonmitotic cells, but they also offered innovative solutions to overcome some of the
intracellular barriers to nonviral gene delivery.
2.4.3.2 Transport of the DNA During Mitosis
The second mechanism of the DNA entry into the nucleus involves association of the
DNA with the nuclear material on breakdown of the nuclear envelope during mitosis.
The comparatively higher transfectability of dividing cells to the dormant nondividing
cells suggests that plasmid DNA enters the nucleus preferentially upon the disassem-
bly of the nuclear envelope during mitotic cell division [182-184] . This hypothesis
is supported by the significant size of plasmid DNA (2-10 MDa), which makes it
unlikely that nuclear entry occurs by passive diffusion in postmitotic cells. These
findings have been observed during several studies carried out with both cationic
lipid and polymer-mediated gene delivery, in which transfection efficiencies for cells
at various stages in the cell cycle were analyzed [182-185] . DNA has been typically
observed as localized in the perinuclear region of the cell, where it presumably awaits
breakdown of the nuclear membrane [184,186] .
Considering degradation of the DNA against the cytosolic nucleases, the enhanced
nuclear delivery of DNA when complexed with the cationic polymer and peptides as
PEI—and to a lesser extent, polylysine—may be because these vectors remain bound
to DNA in the cytoplasm, where they are trapped prior to mitosis [150] . However,
the same observation is not true for cationic lipid-based delivery system. This fact
was demonstrated by the study of microinjection of cationic lipid-DNA complexes
into the cytoplasm, which should also protect DNA from degradation by nucleases; it
does not result in a significant amount of nuclear DNA uptake, thereby suggesting an
additional significant role of cationic polymers in the nuclear delivery of DNA [150] .