Synthetic Dicer-Substrate siRNAs as Triggers of RNA Interference - RNA Interference from Biology to Therapeutics - page 46

[ 76 ]. Bacterial lipopolysaccharide (LPS) binds the receptor TLR4 and triggers
release of a number of pro-inflammatory cytokines, including large amounts of
TNF-a by macrophages. The “cytokine storm” induced by LPS can lead to hypoten-
sion, shock, and death. One early sign of the severity of this shock state is hepatic
necrosis. Mice were administered the anti- Tnf DsiRNA complexed with TransIT-
TKO™ as a single 5 mg dose (0.25 mg/kg) in 200 mL volume by IP injection imme-
diately prior to injection of the low dose LPS/D-GalN cocktail. Downregulation of
TNF-a production by peritoneal mononuclear cells following this treatment reduced
the magnitude of liver damage and delayed mortality. This study again demon-
strated the ability to achieve systemic effects in an inflammatory disease state using
IP administration of a DsiRNA targeting a cytokine expressed in macrophages.
Howard, Kjems, and colleagues employed this same strategy to treat collagen-
induced arthritis (CIA) in mice [ 77 ]. In this case, a novel chitosan-based nanopar-
ticle delivery system was employed [ 86 ]. The chitosan nanoparticles employed in
this study exhibit a net positive charge that may lead to serum-induced aggregation
after IV injection but are ideal for use with IP injection and are efficiently taken up
by peritoneal macrophages. TNF-a secreted by macrophages is an important factor
in the development and pathophysiology of inflammatory arthritis, and anti-TNF-a
antibodies are presently employed to treat rheumatoid arthritis. Mice were immu-
nized with an anthrogen-CIA collagen emulsion and displayed the onset of arthritis
within 28-35 days postinjection, at which point DsiRNA treatments were initiated.
Mice received either 5 mg of chitosan-complexed unmodified anti- Tnf DsiRNAs or
control DsiRNAs (0.25 mg/kg) or 2.5 mg of chitosan-complexed 2¢ -OMe-modi fi ed
anti- Tnf DsiRNA (0.125 mg/kg) in 200 mL volume by IP injection on days 1, 3, 5,
7, and 9. Arthritis scores were measured, and joints were examined by histopathol-
ogy. Suppression of TNF-a by IP administration of an anti- Tnf DsiRNA reduced
joint swelling, and mice receiving this treatment showed minimal erosion of the
articular surfaces of affected joints, while control mice showed severe cartilage
destruction. Overall, use of 2¢-OMe-modified DsiRNAs showed the most effective
biological responses.
The same group used the chitosan-based nanoparticle delivery system to treat
radiation-induced fibrosis (RIF) in mice using the IP administration route [ 78 ] . RIF
arises from damage and scarring within tissue exposed to high levels of ionizing
radiation and is characterized by reduced mobility, strictures, pain, and even tissue
necrosis. Morbidity from RIF can limit the dose of radiation that can be adminis-
tered in the treatment of various cancers, and methods to reduce or prevent RIF
would have significant value. There is some evidence to suggest TNF-a plays a
potentially significant role in this disease. The hind limbs of mice were given a
single 45-Gy dose of gamma radiation. Chitosan-complexed unmodified control or
anti- Tnf DsiRNAs were administered at a dose of 5 mg (0.25 mg/kg) in 200 m L
volume by IP injection for a varying periods of time, given 2 days prior to irradia-
tion, 1 day post-irradiation, and biweekly thereafter. Mice receiving anti- Tnf
DsiRNA therapy for <3 weeks all developed RIF, whereas none of the mice that
received continuous therapy for 3 weeks or longer developed RIF. All mice
that received the scrambled control DsiRNA developed RIF. This work suggests