Lipidoids: A Combinatorial Approach to siRNA Delivery - RNA Interference from Biology to Therapeutics - page 141

The translation from in vitro experiments to proof-of-concept in vivo studies was
rapid. The report of successful silencing in vivo in 2002 [ 6 ] served as an exciting
advance in the field, and the first demonstration of RNAi-mediated disease inhibi-
tion in an animal model was reported only 1 year later [ 7 ] . In 2004, the fi rst example
of therapeutic silencing of an endogenous gene following systemic administration
of cholesterol-modified siRNAs was described [ 8 ]. More advanced systems would
soon follow, and the utility of RNAi as a platform technology was validated in non-
human primates (NHP) within 2 years [ 9 ]. Importantly, some of the safety concerns
attributed to shRNA-mediated gene silencing [ 10 ] were not found to be relevant to
siRNA-mediated gene silencing. siRNA enters the RNAi pathway downstream of
nuclear export and Dicer processing, so it does not interrupt the endogenous
microRNA pathway [ 11 ]. Saturation of Ago2 loading is possible in vitro but is
unlikely to be of concern in vivo, where efficient delivery remains a challenge.
Clearly, the ability to deliver siRNAs in vivo would enable a fundamentally new
way of treating disease, catalytically preventing protein production rather than stoi-
chiometrically inactivating aberrant proteins following their translation, as tradi-
tional pharmacological methods do.
There are, however, several significant barriers to entry. siRNAs are large, poly-
anionic molecules that do not readily cross the hydrophobic, tightly packed cell
membrane, so carriers must be used. These carriers must protect the drug payload
from degradation, filtration, and phagocytosis in the bloodstream; facilitate trans-
port across the vascular endothelial barrier; allow for diffusion through the extracel-
lular matrix; and promote cellular uptake as well as cargo release into the cytosol
[ 12 ]. While extremely efficient mediators of DNA delivery, viruses do not deliver
synthetic siRNA and can result in insertional mutagenesis when delivering shRNA,
whose expression levels are difficult to control. For these reasons, synthetic deliv-
ery systems have been developed. Polycationic polymers and lipids are often used
because they can electrostatically bind and condense the polyphosphate backbone
of nucleic acids to form nanometer-sized particles that enhance stability and cellular
uptake. A novel class of lipid-like compounds, termed “lipidoids,” was developed in
order to expand the scale and diversity of systemic siRNA delivery systems. This
chapter will focus on the synthesis, screening, and application of these molecules.

7.2 Rational Versus Combinatorial Approaches

Though lipid-based formulations for systemic siRNA delivery represent one of the
most promising near-term opportunities for the development of RNAi therapeutics
[ 13 ], the present collection of delivery materials and their diversity remain limited.
The central obstacle to the expansion of scale is the slow, multistep syntheses
required to create cationic lipids. The result of this laborious and time-consuming
approach is a low-throughput, iterative process.
When performed rigorously, this directed approach can be useful for interrogat-
ing the effects of minor differences on functional properties toward the elucidation