Biomedical Engineering Reference
In-Depth Information
tissue macrophages, whose physiological function is to clear foreign pathogens
and to remove cellular debris and apoptotic cells.
29
Tissue macrophages are
most abundant in the liver (where they are called Kupffer cells) and the spleen,
tissues that also receive high blood flow and exhibit a fenestrated vasculature.
Thus, it is not surprising that these organs accumulate high concentrations of
siRNAs following systemic administration.
30
Four hours after hydrodynamic
i.v. tail vein injection with fluorescently labeled siRNA, a strong fluorescent
signal was noted in the liver, spleen, and bone marrow. Similarly, siRNA
uptake after standard i.v. tail vein injection or i.p. injection was noted in the
liver, spleen, kidney, and bone marrow at 4 h, but the overall signal was
weaker.
31
Even after different chemical modifications, high concentrations of
phosphodiester (PO), phosphorothioate (PS), 29-OMe, and locked nucleic acid
(LNA)-modified siRNA duplexes were found in large nucleated cells within the
red bone marrow,
32-34
in the red pulp of the spleen,
32,33,35
and in the Kupffer
cells of the liver.
36
These findings suggest that RES-mediated uptake of siRNA
plays an important role in its tissue distribution.
siRNAs can generate off-target effects and can lead to unanticipated
phenotypes that complicate the interpretation of the therapeutic benefits of
siRNA, including siRNA-induced sequence-dependent regulation of uninten-
ded transcripts through partial sequence complementarity to their 39-UTRs, as
well as widespread effects on microRNA processing and function through
saturation of the endogenous RNAi machinery by exogenous siRNAs.
37
Off-
target effects are used in RNAi screens to identify novel components of signal
transduction pathways in a variety of organisms. All siRNA hits, whatever
their intended direct target, reduce the mRNA levels of two known upstream
pathway components, the TGF-b receptors 1 and 2 (TGFBR1 and TGFBR2),
via micro (mi)RNA-like off-target effects. The scale of these off-target effects is
remarkable, with at least 1% of the sequences in the unbiased siRNA library
having measurable off-target effects on one of these two genes.
38
Transfection
of small RNAs can globally perturb gene regulation by endogenous miRNAs.
Targets of endogenous miRNAs are expressed at significantly higher levels
after specific siRNA transfection, consistent with impaired effectiveness of
endogenous miRNA repression, which results in unexpected gene expression
changes.
39
Another challenge for siRNA therapy is immune stimulation, as introducing
too much siRNA is known to result in nonspecific events owing to the
activation of innate immune responses.
40
siRNAs can activate the cells of the
immune system and induce the production of cytokines in vitro and in vivo.
41-43
Mammalian immune cells express a sub-family of pattern-recognition
receptors called Toll-like receptors (TLRs) that recognize pathogen-associated
molecular patterns, including unmethylated CpG DNA and viral dsRNA.
44
Several TLRs are involved in the recognition of siRNAs, including TLR3,
TLR7, and TLR8.
45,46
TLR3 is the receptor for dsRNA and TLR3
overexpressed in cultured human embryonic kidney (HEK) 293 cells is capable
of recognizing siRNAs.
47
siRNAs have been shown to activate TLR3 signaling
d
n
4
y
3
n
g
|
8
