Biomedical Engineering Reference
In-Depth Information
9.6
Thinking Outside the Box: Bi-Functional
siRNAs
In addition to improving delivery techniques for RNAi, we have
investigated improving the efficacy of RNAi by combining targeted
siRNA knockdown with local immune activation. The concept
is simple — local immune activation, via triggering of toll-like
receptors, in both the viral and tumour setting may offer more
potent and beneficial therapeutic effects over siRNA alone. Potent
immune activation by dsRNA longer than 30 bp has been clearly
documented and leads to cellular shutdown of protein expression.
Immune activation occurs as a consequence of dsRNA recognition
by the immune system as a common intermediate in many virus life
cycles [64]. Originally, 21 bp siRNAs were thought to be too small to
be recognised by the sensors and receptors of the innate immune
system. However, activation of a robust immune response by
siRNAs has now been well established [65]. To date, innate immune
activation and subsequent cytokine production by siRNAs have been
shown to be predominately mediated by immune cells and caused
by recognition via immune sensors of viral infection. The best recog-
nized receptors and sensors of siRNAs are the toll-like receptors
3-7 and 8, retinoic acid inducible gene 1 (RIG-1), melanoma
differentiation associated gene 5 (MDA-5), and dsRNA dependent
protein kinase (PKR). Each receptor is activated by different motifs
and structures in siRNAs. For example, PKR is activated by long (<30
bp) dsRNA and siRNAs [66], while RIG-1/MDA-5 are activated by a
5
-triphosphate [67]. As these molecules form part of the normal
anti-viral response, siRNA activation of these receptors and pathways
may be advantageous in the treatment of virally induced diseases.
Since it was originally believed that siRNAs were too small to
be detected by the immune system, off-target immune activation
effects were often poorly controlled in early RNAi studies and many
results may have been due more to such activation than RNAi-
mediated gene silencing. This was shown to be the case by Tekmira
Pharmaceuticals, which demonstrated that in a murine model of
influenza infection, the reported anti-viral activity of siRNA was not
due to RNAi silencing, but was primarily caused by siRNA immune
stimulation [68]. The misinterpretation of anti-viral activity was
attributed to the inadvertent low immunogenicity of the control
siRNA compared to the anti-influenza siRNA. Indeed, this control
siRNA had been used by a number of groups [49, 69-72], and thus
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