Challenges and Opportunities in Gene Delivery - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 52

delivery, comparatively diminished transfection efficiency has been observed, sugge-
sting significant influence of the barriers encountered during transgene expression.
After systemic delivery of nucleic acids by intravenous and intramuscular injection,
various barriers in the extracellular milieu have been observed, obstructing the delivery
of nucleic acids to the surface of the desired cellular target. These extracellular barri-
ers include physical properties of DNA such as size, shape, and charge influencing the
biodistribution and cell permeation; DNA degradation by nuclease; and hepatic uptake
and metabolism, thus limiting the cell access and, effectively, transgene expression
[22,43-45] .
Plasmid DNA is a macromolecule having a molecular weight of about 2500 kDa
or more, with a phosphate backbone having a strong anionic charge. Such a size and
charge significantly alters the biodistribution and cell uptake of the plasmid DNA once
injected as intravenous injection. The supercoiled, open circular or linear nature of
the plasmid DNA affects the cell uptake, with open circular DNA showing compara-
tively more transgene expression than the linear and supercoiled counterpart [47] . The
transgene expression is also hindered by the metabolic or enzymatic instability against
serum nuclease. Abundantly available extracellular and serum nuclease in the extracel-
lular environment rapidly degrades the plasmids after intravenous and intramuscular
administration [18,22,42] . However, the problem of degradation has been resolved to
an extent by condensing the DNA with a variety of polycations (cationic lipids and
polymers—lipoplexes or polyplexes) and by complexing DNA with cationic polymers
thereby protecting it from degradation against serum nuclease [18,48-50] . The effect
of serum nuclease on DNA degradation and transgene expression can be explained by
the fact that, more transgene expression is observed even after naked DNA injection
into skeletal muscles where comparatively lower nuclease levels are observed [51] .
The mechanism of degradation of DNA by serum nuclease is dependent on the pres-
ence of divalent cations, such as calcium in serum, which activate the enzyme for DNA
degradation, preferably by hydrolysis [52,53] . Use of metal ion chelators to stop the
DNAse-mediated DNA degradation has also been mentioned in various molecular biol-
ogy protocols, confirming the role of divalent cations in activity of DNAse [54] .
Another barrier to transgene expression is thought to be the large size of the circu-
lating plasmid DNA, which hinders its access to cells except for blood cells, endothe-
lial cells, and parenchymal cells in the liver and spleen. Generally, the molecules with
size greater than 5 nm diameter do not cross the capillary endothelium easily because
of tight endothelial junctions, and they are cleared from circulation in due time.
However, some organs such as the liver and spleen, and leaky vasculatures such as
solid tumors have demonstrated entry of the macromolecules, including DNA, with or
without carriers, having size up to 200 nm, thereby allowing transgene expression [55] .
Apart from these barriers, DNA is found to be hepatically cleared with high first-
pass metabolism. The uptake of naked DNA occurs by nonparenchymal (probably
Kupffer) cells, following the interaction of DNA with scavenger receptors for polyan-
ions [56] , thus suggesting the need to neutralize the anionic charge of DNA to avoid
hepatic uptake and metabolism. The negatively charged DNA is also less permeable
to the negatively charged cell membrane, thus having a diminished activity and trans-
gene expression.