Challenges and Opportunities in Gene Delivery - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 67

the nuclear envelope at fairly high surface densities (3000-4000 per nucleus) and
exist in at least two conformational states [166] . Molecules smaller than or approxi-
mately 40 kDa can diffuse passively through the NPC. Macromolecules larger than
60 kDa either contain a specific targeting signal, the NLS, or associate with other
polypeptide(s) incorporating NLS to traverse the NPC in an energy-dependent
manner [167] . The dynamic behavior of the NPC indicates that specific transport
signals provoke considerable conformational change in the NPC. The closed state
permits the passive diffusion of molecules of 9 nm in diameter, whereas the open
state facilitates transport of particles up to 26 nm [168,169] . This open state could
certainly accommodate the “threading” of supercoiled plasmid through the nuclear
pore, but not passage of typical nonviral gene delivery complexes. In a few cases,
collapsed particles of 30 nm have been produced, which could presumably enter
the nucleus by this mechanism [170-172] . This conformational change provides a
plausible explanation for the ability of the NPC to permit the translocation of sub-
strates as large as 25-50 MDa [173] .
Enhancement in DNA transport through nuclear pores is possible by modifica-
tion of the DNA sequence to be inserted to include binding sites for karyophilic pro-
teins that facilitate the nuclear pore transport. This phenomenon was first observed
in plasmids containing the SV40 enhancer region, which binds a variety of transcrip-
tion factors [165] . This approach was further utilized by including tissue-specific
promoter sequences in DNA sequences that may interact with the specific transcrip-
tion factors in the cytoplasm, specifically of those tissues, which are then are trans-
ported to the nucleus through the NPCs. The concept was efficiently applied by Dean
et al. during transfection of the smooth muscle cells by the DNA, after incorporating
the promoter for the smooth muscle gamma actin transcription factor. The promoter
helped to effectively transport the DNA into the nucleus of smooth muscle cells and
produced gene expression, whereas little DNA was trafficked to the nucleus of CV-1
cells demonstrating tissue-specific DNA expression [174] . Therefore, this approach
provides the potential to increase the amount of transgene trafficked to the nucleus
while also allowing tissue-specific targeting.
Another widely reported approach to enhance the nuclear translocation and
expression of DNA involves covalent modification of DNA by attachment of NLSs
[36] . The quantitative attachment of NLSs to the DNA must be optimized, with vary-
ing transfection efficiency observed after using varying quantities of NLSs like prot-
amine sulfate, per mole of the DNA, and cationic lipid used as a vector [175,176] .
An optimal degree of labeling with the NLSs was apparent in these studies, with too
many signals per DNA copy thought to inhibit translocation by simultaneously inter-
acting with multiple nuclear pores. The use of peptide nucleic acids to noncovalently
attach nuclear localization signals by the formation of site-specific DNA triplexes
has also been reported [176] . An interesting possibility is that positively charged
regions of gene delivery complexes, especially the cationic polymers and peptides,
could themselves serve as NLSs [175] .
The NLSs attached to the DNA to be transported are recognized by the importin-a
adapter on the NPCs, which in turn forms complex via the importin-h-binding domain
with importin-h. Following the nuclear uptake of the complex, association of Ran-GTP