Synthetic Dicer-Substrate siRNAs as Triggers of RNA Interference - RNA Interference from Biology to Therapeutics - page 44

2.3 Studies Using DsiRNAs In Vivo

Many studies have been published that employ siRNAs in animals. The scientific
issues involved in conducting in vivo RNAi studies in mammals and overviews of
significant articles in this rapidly growing field have been well covered in recent
reviews [ 73, 74 ]. Some recent successes using DsiRNAs in vivo will be discussed
below. The in vivo studies discussed herein are summarized in Table 2.1 .
Like all classes of synthetic dsRNAs, delivery is the most significant hurdle to
widespread use of DsiRNAs in vivo. While the systemic effects associated with
intravenous injection are often desirable, local administration is usually easier to
perform and suffers from fewer toxicity issues. Several studies have been performed
with DsiRNAs using local delivery, including intraperitoneal (IP) and intrathecal
(IT) routes of administration. Amarzguioui and colleagues described methods to
deliver DsiRNAs using an inexpensive commercially available cationic lipid in mice
via IP injection [ 85 ]. It was observed that the lipid TransIT-TKO™ was particularly
effective in delivering DsiRNAs into macrophages, both in cell culture and in vivo.
The peritoneal cavity is a reservoir for monocyte lineage cells, and IP injection
offers an opportunity to easily introduce DsiRNA reagents into this cell population
via local administration. Immune cells like macrophages are mobile: the transfected
macrophages can be recruited to sites of inflammation outside of the peritoneum,
thereby achieving systemic effects using a local administration strategy.
Lundberg, Cantin, and colleagues employed this strategy to suppress production
of tumor necrosis factor alpha (TNF-a) by macrophages during acute herpes sim-
plex virus (HSV) infection in the mouse central nervous system (CNS) [ 75 ] . Studies
were performed in C57BL/6, a strain of mice that normally survives herpes enceph-
alitis (HSE). TNF-a is an important factor in the resistance of this strain to HSE,
and C57BL/6 mice that are double knockouts for the two known TNF receptors
(p55 −/− , p75 −/− ) show increased fatality following HSE. Mice were administered the
anti- Tnf DsiRNA DsiRNA complexed with the cationic lipid TransIT-TKO™ as a
single 2 mg dose (0.1 mg/kg) in 200 mL volume by IP injection immediately prior
to infection with HSV, then received five additional doses of 4 mg (0.2 mg/kg) on
days 1, 2, 4, 6, and 8 [for a total of six doses with a total cumulative dose of 22 m g
(or 1.1 mg/kg) of the DsiRNA]. Surprisingly, suppression of TNF-a production
using an anti- Tnf DsiRNA resulted in significantly increased mortality rates, sug-
gesting that the presence of TNF-a somehow alters the pathophysiology of HSE
even in the absence of the two known TNF receptors. The mechanism of this unex-
pected observation remains under investigation. In this case, it is not believed the
IP administered DsiRNA directly entered the mouse CNS. Rather, the DsiRNA
were taken up by macrophages, which were subsequently recruited by the
inflammatory process ongoing in the brain due to the HSV infection, thereby
achieving CNS effects from IP administration of a compound that normally cannot
cross the blood brain barrier (BBB).
Lundberg, Cantin, and colleagues later used this same system (IP injection of
anti- Tnf DsiRNA complexed with TransIT-TKO™ to transfect macrophages) to
alter the course of fatal hepatic necrosis during endotoxin-induced “toxic shock”