Synthetic Dicer-Substrate siRNAs as Triggers of RNA Interference - RNA Interference from Biology to Therapeutics - page 48

cells, such as plasmacytoid dendritic cells and B cells. Mice also express TLR9 on
myeloid dendritic cells and monocytes. Like the RNA-binding receptors TLR3, 7,
and 8, TLR9 primarily resides in the endosomal compartment. A DsiRNA targeting
Stat3 was conjugated to a phosphorothioate-modified single-stranded DNA sequence
termed CpG 1668, a known potent class B TLR9 agonist [ 87 ] . Stat3 expression is
increased in some tumors where it promotes cell division and tumor growth. It also
reduces local immune responses to the tumor, thereby further aiding tumor survival.
Thus, suppressing Stat3 could be beneficial and reduce tumor cell survival by sev-
eral mechanisms. The anti- Stat3 +CpG DsiRNAs were taken up by cells into the
endosomal compartment via a TLR9-independent mechanism. Cells lacking TLR9
did not show any gene suppression effects. For cells expressing TLR9, some frac-
tion of the material escaped into the cytoplasm and led to suppression of Stat3 . This
reagent was used to treat mice bearing subcutaneous implants of syngeneic B16
melanoma cells. Direct peritumoral local injections of the anti- Stat3 +CpG DsiRNAs
were performed in the tumor nodules at a dose of 0.78 nmol per injection (20 m g, or
~1 mg/kg). After three daily injections, significant regression of the B16 tumor
implants was observed. Next, B16 tumor cells were injected IV, and the growth of
pulmonary nodules was measured over time (an established method to mimic the
behavior of metastatic melanoma). The anti- Stat3 +CpG DsiRNAs were adminis-
tered IV at a dose of 0.78 nmol per injection (20 mg, or ~1 mg/kg). The intravenous
injections were given every other day for 2 weeks and led to a significant reduction
of both the size and number of B16 implants detectable in the lungs of mice receiv-
ing the anti- Stat3 therapy but not in the control mice. Another example for ligand-
mediated delivery, Nishina and colleagues used direct IV injection of a highly
modi fi ed anti- Apob DsiRNA conjugated to vitamin E (a-tocopherol) to reduce
expression of apolipoprotein B in liver and reduce serum cholesterol levels [ 63 ] .
Refer to discussion in Sect. 2.2.2.1 for additional details.
Aptamers are highly structured nucleic acid molecules which form conforma-
tions that can bind a target ligand with high affinity and specificity, much like anti-
bodies. Aptamers can be conjugated to other compounds to facilitate targeted
delivery. For more information, see recent review articles by Thiel [ 88 ] , Zhou [ 89 ] ,
and Syed [ 90 ]. Aptamers have been employed to facilitate delivery of DsiRNAs
both in vitro and in vivo.
Zhou and colleagues developed an aptamer specific for the HIV-1 gp120 protein
expressed on the surface of HIV-infected cells [ 91, 92 ] . This anti-gp120 aptamer
was fused to a DsiRNA specific for the HIV tat/rev gene. The chimeric aptamer-
DsiRNA molecule was synthesized by in vitro transcription (IVT) using 2¢ -F pyrim-
idine bases to improve nuclease stability, thereby permitting use of the fusion
construct in serum without the necessity for additional protection. The aptamer
component was shown to target HIV-infected cells and facilitate DsiRNA uptake.
Following uptake, the aptamer and DsiRNA are separated by Dicer processing to
liberate an active 21-nt siRNA that directs suppression of the HIV tat/rev gene. Neff
and colleagues studied use of the gp120-aptamer anti- tat/rev DsiRNA fusion mol-
ecule to suppress HIV infection in a humanized mouse immune system [ 83 ] . The
humanized Rag2 −/− g c −/− (RAG-hu) mouse is a rodent model system that allows the
study of sustained chronic HIV infection. Over time, infected mice show declining