Antisense Oligonucleotides and RNA Interference - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 337

agent in such polymer-AS ODN complexes. Also endo-osmolytic agents like chlo-
roquine and a higher concentration of other osmotic agents like glycerol, sucrose, or
polyvinyl pyrrolidone (PVP) have been shown to aid osmolysis of endosomes.
7.4.2.3 Nuclear Localization
Following endosomal escape, release from the cationic complex of siRNA in cytoplasm
and nuclear localization in AS ODNs is required for interaction with target mRNA to
inhibit related protein expression. Addition of an anionic lipid can displace negatively
charged siRNA or AS ODN from the cationic lipid-polymer-oligonucleotide complex
[66,67] . This release is attributed to the multivalent nature of the anionic lipid and the
electrostatic and hydrophilic-hydrophobic interactions of the lipids. This lipid mix-
ing results in charge neutralization, allowing the diffusion of the cationic lipids away
from the oligonucleotide. Thus, the anionic lipid competes with the anionic oligonucle-
otide for the cationic lipid, displacing the oligonucleotide. In the cationic polymer-AS
ODN complex, cationic polymers like polyethyleneimine (PEI) or poly-L-lysine (PLL)
accelerates nuclear localization by preventing cytosolic degradation of the complex.
The extent and duration of the oligonucleotides' response depends upon the step limit-
ing the rate of cellular uptake, intracellular trafficking, endosomal release, and nuclear
entry. This step can be controlled by the conjugation of cell-penetrating signal peptides,
endosomal release signal peptides, or nuclear localization signal peptides to the oligo-
nucleotides. However, siRNA polyplexes with reducible polymers, such as polyethyl-
eneglycol-poly-L-lysine (PEG-PLL) block copolymers with disulfide crosslinking, are
preferred for cytosol-specific degradation, to release siRNA in cytoplasm to act on tar-
get mRNA in cytosol [68] .
7.4.2.4 Inhibition of Protein Expression
After nuclear localization, the AS ODN must bind to the complementary mRNA,
thereby downregulating the related protein expression. The transfection efficiency of
these cationic lipid-polymer-oligonucleotide complexes depends on the lipid/poly-
mer-to-oligonucleotide ratio [57] . Hence, the lipid/polymer-to-oligonucleotide ratio
must be optimized to achieve maximum inhibition of mRNA expression. Inclusion of
tissue-specific promoters can be tried to inhibit mRNA expression within the thera-
peutic window [69,70] . Formulation with the appropriate concentration decided after
a sound understanding of the pharmacokinetics and biodistribution of a particular
antisense molecule and the appropriate modifications to overcome undesired barriers,
can provide further value to antisense therapeutics.
7.4.3 Immunological Barrier
The body has an inbuilt mechanism to fight against invading foreign bodies such as
pathogens. This resistance is conferred by two distinct mechanisms, namely, innate
immunity and adaptive immunity, and presents a major barrier to intracellular AS
ODN delivery. An individual is born with innate immunity, which is mediated by