Challenges and Opportunities in Gene Delivery - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 66

of cell-localized polyanions. In this idea, it is envisioned that cationic complexes
are passed from proteoglycans to cytoplasmic-spanning polyanions of increasing
affinity, such as tubulin (microtubules) and actin (microfilaments), to the nucleic
acid-rich nuclear region [43] . Although the mechanism by which cationic polymers
still bound to DNA in the cytoplasm facilitate increased transgene expression is still
unclear, enhanced diffusion, because of the condensed nature of the complex, and
protection of DNA from cytosolic nucleases are probably at least partially involved.
2.4.3 Nuclear Transport of Plasmid DNA
The nuclear envelope represents a most formidable barrier to the nuclear entry of
plasmid DNA in the cells, particularly in nondividing cells. However, the delivery of
DNA to the nucleus must occur for the efficient transgene expression to take place.
The inefficient nuclear uptake of plasmid DNA from the cytoplasm was recognized
more than 30 years ago. Comparison of the transfection efficiency of plasmid DNA-
encoding thymidine kinase, introduced either into the cytosol or the nucleus, showed
that not more than 0.1-0.001% of the cytosolically injected plasmid DNA could be
transcribed [163] . Similar results were obtained more recently by injection of the
-galactosidase reporter gene detection of radioactive or fluorescent plasmid DNA
[150,156] . These studies demonstrated very low probability of intact plasmids arriv-
ing in the nucleus by a passive process. The literature also describes the transport
of macromolecules, including plasmids, to the nucleus as a very inefficient process,
and until date, the attempts to achieve the efficient nuclear uptake of the macromole-
cules by passive process as well as by active transport through the nuclear pores have
achieved limited success.
The mechanism of DNA nuclear translocation and association of DNA with the
delivery system until it reaches the nucleus is still not fully understood but appears
to depend on the type of delivery vehicle employed, such as cationic lipid or poly-
mer. At least three possible routes exist for DNA transport alone or along with the
carrier system to the nucleus. The DNA can pass into the nucleus through nuclear
pores by passive transport or active transport with the help of a suitable promoter
or a nuclear localization sequence (NLS); it can become physically associated with
chromatin during mitosis when the nuclear envelope breaks down or it could traverse
the nuclear envelope. Of these three possibilities, the latter seems the least likely and
as of yet has no experimental support.
2.4.3.1 Transport of the DNA Through the Nuclear Pore
Nucleocytoplasmic transport of macromolecules as DNA, RNA, proteins, peptides,
and so on, through the nuclear membrane is a fundamental process for the meta-
bolism of mammalian cells. The cytonuclear trafficking of proteins and ribonucleo-
proteins is controlled by the NPCs by forming an aqueous channel through the nuclear
envelope [164] . Transport of plasmid DNA through the nuclear pores has been impli-
cated in numerous studies, particularly when the plasmid contains specific sequences
recognized by host cell transcription factors [165] . Nuclear pores are embedded in