Lipidoids: A Combinatorial Approach to siRNA Delivery - RNA Interference from Biology to Therapeutics - page 149

half-life in serum from ~15 min to >24 h in vitro. The formulation was monitored
for 5 months following storage at 4, 25, or 37°C, and particle size distribution and
siRNA entrapment were unchanged over this time course. No loss in efficacy was
detected for a batch of particles that had been stored for 20 months at 4°C. This lead
formulation efficiently accumulated in the liver (>90% injected dose in 1 h) and
induced fully reversible, long-duration gene silencing—up to 4 weeks—without
loss of activity following repeated administrations of modified siRNA [ 31 ] .
The pharmacodynamics of this systemically administered siRNA formulation were
assessed using a mouse that expresses luciferase ubiquitously [ 34 ] . Since the entire
animal emits bioluminescence in the presence of substrate, the mice must be eutha-
nized and organs must be harvested prior to analysis. Nonetheless, this mouse strain
is a useful tool for the rapid screening and optimization of siRNA formulations,
particularly for tissue-targeted delivery systems. Importantly, it was confirmed that
the formulation is relevant to multiple hepatocyte-expressed target genes, as apolipo-
protein B (ApoB) was also effectively silenced [ 11 ]. Furthermore, the observed effects
were dose dependent and specific compared to mismatch control siRNA [ 18 ] .
The specificity of siRNA can be lost when it induces a general innate immune
response, which sometimes occurs when unmodified siRNAs are used [ 35 ] . siRNAs
can interact with toll-like receptors (TLRs) in a structure- and sequence-dependent
manner [ 36 ]. The inclusion of chemically modified nucleotides, such as of 2¢ - fl uoro
pyrimidines and 2¢ - O -methyl purines, not only increase nuclease resistance [ 37 ] but
also abrogate the interferon response [ 38 ], suppressing interaction with TLR7 and
TLR8 [ 39 ]. In some instances, such as antiviral applications, the combination of
specific target gene silencing (mediated by siRNA) and general immunostimulation
(mediated by so-called immunostimulatory RNA, or isRNA) can confer synergistic
outcomes [ 40 ] .
Interestingly, it was found that not only is the structure of the siRNA critical to
the modulation of innate immunity, but also the lipidoid formulation is important
[ 41 ]. The formulation of the lipidoid 98N 12 -5(1) was optimized to maximize the
immunostimulatory capacity of unmodified siRNA in a mouse model of influenza.
Specifically, lyophilization of the particles resulted in increased particle size, which
is believed to have led to increased uptake by interferon-producing resident plasma-
cytoid dendritic cells (cf., hepatocytes) in the liver. This formulation method
enhanced the antiviral effects of the siRNA payload, providing efficient prophy-
lactic inhibition of influenza A in mouse lungs. Significantly, lipidoids can also be
applied to afford highly specific gene knockdown in the absence of an innate
immune response, and this has been demonstrated in several disease models.

7.6 Applications

Lipidoids were first used to address targets relevant to hypercholesterolemia. siRNA
was delivered efficiently to the liver to silence proprotein convertase subtilisin/
kexin type 9 (PCSK9) [ 42 ] and ApoB [ 18 ] in non-human primates (NHP). The
silencing of PCSK9 increases low-density lipoprotein (LDL) receptor protein levels