Challenges and Opportunities in Gene Delivery - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 65

Diffusion of DNA in the cytoplasm has been found to be substantially less than that
observed in dilute solution [155] . For DNA 2000 base pairs in length, the diffusion
coefficient in the cytosol is 1% of that in water, suggesting a substantial diffusional
barrier. This decreased mobility has been ascribed to molecular crowding of the
plasmid [155] but may also reflect an increased viscosity of the cytoplasm and bind-
ing interactions of DNA with intracellular components. As expected, the diffusion
coefficient of DNA in the cytoplasm is inversely related to the size of the plasmid,
suggesting smaller plasmids may be more desirable [155] . No evidence has been
reported demonstrating active transport of DNA from the cytoplasm to the nucleus.
Consistent with the notion that lateral diffusion may limit nuclear entry, microin-
jection of plasmid DNA into the proximity of the nucleus or decreasing the size of
the expression cassette led to significant enhancement of the transfection efficiency
[156,157] . Because the mobility of DNA is inversely proportional with the size of the
DNA-polycation complex, it is reasonable to assume that the faster mobility of con-
densed DNA could account, at least in part, for the enhanced transfection efficiency
of the PEI-complexed plasmid DNA [150] . Vesicular delivery of the synthetic vector
to the perinuclear endosomal compartment would be ideal to minimize the cytoplas-
mic exposure time and diffusional distance of plasmids prior to their nuclear uptake
can occur.
In addition to the considerable diffusional barrier for DNA in the cytosol, the pres-
ence of calcium-sensitive cytosolic nucleases poses a significant metabolic barrier as
well [158,159] . Microinjection of DNA into the cytoplasm results in significant deg-
radation of the DNA, with a half-life of 50-90 min [159] . The microinjected DNA
monitored and estimated by the fluorescent in situ hybridization (FISH) technique
by time-dependent decay kinetics of the FISH signal revealed that 50% of the DNA
is eliminated in 1-2 h from HeLa and COS-1 cells and in 4 h from C2C12 cells and
myotubes [159] . The fast turnover rate of microinjected DNA was independent of the
copy number (1000-10,000 plasmid/cell) and the conformation (linearized, super-
coiled versus open circular; single- versus double-stranded) of the plasmid. Cytosolic
elimination of plasmid DNA could not be attributed to cell division, because compa-
rable degradation was observed in cell cycle-arrested cells thus indicating metabolic
instability of DNA contributing to the low efficacy of gene transfer [159] .
In vitro studies have demonstrated that DNA-polycation complex formation dra-
matically increases the nuclease resistance of plasmid DNA [150,160,161] . Consistent
with the diminished nuclease susceptibility of complexed DNA, encapsulation of
microinjected plasmids into stabilized lipid particles has shown to delay the degrada-
tion of DNA more than three fold [159] . These results suggest that faster diffusional
mobility as well as augmented nuclease resistance account for the enhanced nuclear
targeting efficiency of the PEI-condensed plasmid DNA [150,159] . Similar phenom-
ena could explain the more than 10-fold improved transfection efficiency of microin-
jected DNA nanoparticles (containing lysine polymers substituted with polyethylene
glycol) as compared with naked DNA [74,162] . These findings confirm the fact
that the relatively rapid metabolic turnover of plasmid DNA in the cytosol imposes
an additional impediment to the nuclear translocation of DNA. It is hypothesized
that the transport of these positively charged complexes is mediated by a gradient