Challenges and Opportunities in Gene Delivery - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 60

2.4 Intracellular Barriers for Naked DNA and DNA
Delivery Systems
After successfully overcoming the extracellular barriers, the DNA-polycation com-
plex enters the desired cells by endocytosis. However, it must still overcome more
critical intracellular barriers to achieve the desired transgene expression. The various
intracellular barriers that must be overcome by the DNA-polycation complexes are
shown in Fig. 3.3 .
2.4.1 Endosomal Escape of DNA from the DNA-Polycation Complex
The endosomal escape of the DNA into the cytosol has been one of the major barriers
to efficient gene delivery, and various approaches have been tried to overcome this
barrier. After receptor-mediated internalization of lipoplexes and polyplexes through
phagocytosis, the vectors are fused with the cytosolic organelles to form early endo-
somes, which are characterized by their DNA-degrading enzyme contents, an acidic
pH around 5.5-6, and their location near the cell wall. The early endosomes are of
two types. One acts during redistribution of the material from the cell surface to cel-
lular organalles (hence, these are called sorting endosomes); the other type is called
a recycling endosome and is thought to be responsible for the return of internalized
material to the cell surface [120] . The internalization, endosomal transport, and redis-
tribution to the cell surface can be best explained by the adenovirus, which, after rapid
internalization, is again partially recycled to the cell surface [121] . However, the recy-
cling of the vector back to the cell surface is dependent on the ability of the vector
to release from the endosome. The nonviral vectors, lacking the ability to efficiently
escape the endosomal compartments, are likely to be recycled back to the cell sur-
face at least partially. Otherwise, the inability of nonviral vectors to escape from the
endosomal compartment may lead to their trafficking via late endosomes to lysosomal
compartments, where the complexes and eventually DNA are enzymatically degraded.
Hence, efficient endosomal escape of the DNA from early endosomes is a vital neces-
sity for therapeutically effective DNA delivery and subsequent transgene expression.
The mechanism of release of DNA from the endosomal compartment varies from sys-
tem to system, such as cationic lipoplexes and polyplexes, and is explained in the fol-
lowing section.
2.4.1.1 Cationic Lipoplexes
The endosomal release of DNA from cationic lipoplexes involves fusion of lipid
components of cationic liposomes and endosomal lipid membranes, which ultimately
leads to membrane disruption and DNA release into cytoplasm [122] . This mechanism
was boosted by the fact that the addition of DOPE (a zwitterionic lipid with a prop-
erty of forming nonbilayer phases with enhanced cell membrane fusion capability)
to the cationic lipid vectors showed enhanced transgene expression [123,124] .