Intracellular Delivery Considerations for RNAi Therapeutics - RNA Interference from Biology to Therapeutics - page 83

specific cells such as hepatocytes [ 60 ]. Recent studies have demonstrated that
cholesterol promotes the fusion with cell membrane and leads to improved intracel-
lular traf fi cking and transfection ef fi ciency [ 61 ] .
Synthetic RNAi vectors can be easily modified with various cell targeting ligands,
including monoclonal antibodies, peptides, small-molecule ligands, and aptamers,
capable of binding to specific cell surface receptors [ 62 ]. Folate is among the most
widely investigated targeting ligands in RNAi therapeutic delivery due to the over-
expression of the folate receptor in cancer cells [ 60 ] . York et al. reported cell-speci fi c
delivery of siRNA using multivalent folate-conjugated block copolymers to obtain
enhanced receptor-mediated endocytosis and cell-specific delivery [ 19 ] .
Aptamers are nucleic acids that can specifically bind to target receptors in specific
cells. This approach is popular for selective delivery of siRNA to target cells [ 60 ] .
It is reported that aptamer-siRNA conjugates are able to selectively enter cells
expressing prostate-specific membrane antigens [ 63 ]. Zhou et al. reported the use of
anti-gp120 aptamers-siRNA conjugates for in vitro silencing of HIV viral replica-
tion to suppress HIV infection [ 20, 21 ] .
Protamine, a short cationic peptide, has been used to link siRNA to F ab fragment
of an HIV-1 envelope antibody to selectively silence gene expression in HIV-infected
cells [ 22 ]. Another reported example of antibody targeting of RNAi includes scFv
antibody used to deliver siRNA to T-cell CD7 receptor [ 23 ] .
Various cell-penetrating peptides (CPPs) such as TAT transactivator protein, penetra-
tin, and transportan have been incorporated into the RNAi delivery vectors to promote
cell uptake and membrane translocation [ 24- 26 ]. Arthanari et al. utilized a fusion pep-
tide of TAT with a membranolytic peptide (LK15) to deliver therapeutic shRNA and
siRNA targeting bcr-abl fusion gene in chronic myeloid leukemia K562 cells [ 27 ] .

4.3.2 Endosomal Escape

It has been suggested that CPPs avoid sequestration of RNAi therapeutics in the endo/
lysosomal pathway and facilitate RNAi cargo delivery directly into the cytosol [ 64 ] .
The endosomal-independent membrane translocation is presumably driven by trans-
membrane potential [ 65, 66 ], with acylation of arginine-rich CPPs being able to pro-
mote direct penetration of the delivery system through the plasma membrane [ 67 ] .
Rydström et al. suggested a CPP-based siRNA delivery system, named CADY, with
some evidence of direct membrane translocation across the plasma membrane [ 28 ] .
Despite the continuing desire and attempts to avoid endo/lysosomal trafficking,
the vast majority of RNAi delivery vectors are internalized by cells via endocytosis.
The intracellular trafficking thus starts in early endosomes and proceeds through the
stage of late endosomes, which are acidified by a vacuolar-type H + ATPase mem-
brane proton pump [ 68 ]. In the absence of an escaping mechanism, RNAi vectors
remain trapped in the endosomes and are subsequently routed to the lysosomes
where they are exposed to lysosomal enzymes and likely degraded [ 69 ] . It is imper-
ative that RNAi vectors either escape or facilitate release of free RNAi therapeutics
from the endosomes before lysosomal degradation.