Synthetic Dicer-Substrate siRNAs as Triggers of RNA Interference - RNA Interference from Biology to Therapeutics - page 50

modification patterns are tolerated, it is likely that individual modification patterns
will be optimized for each clinical candidate rather than using a simple “one size fits
all” universal modification approach.
It is possible to suppress the expression of any desired gene using existing RNAi
methods in vitro. The single greatest impediment to widespread adoption of in vivo
use of these reagents for both research and medical applications is the availability of
effective delivery tools capable of carrying a highly charged anionic RNA duplex
across the cell membrane and deliver it intact in an active form to the cytoplasm
with low toxicity. A wide variety of delivery tools are under development which
may enable use of dsRNAs as drugs. These methods and chemical compositions
have been the subject of numerous excellent reviews in recent years, and the reader
is referred to these sources for additional details [ 88, 95- 105 ]. There is no reason to
believe that 21-nt siRNAs and 27-nt DsiRNAs will show any difference in their
relative efficiency of delivery using cationic lipid or polyplex nanoparticles, so the
same tools should be readily applied across both platforms. The DsiRNA platform,
however, may offer some advantages when used with some other delivery technolo-
gies. For example, it is possible to covalently conjugate carrier molecules to
DsiRNAs so that the modifier is removed by Dicer processing and does not remain
attached to the final mature 21-nt siRNA that actually enters RISC (e.g., at the 3¢ -
end of the sense strand, see Fig. 2.5 ). This may offer an advantage for using DsiRNAs
when employing delivery tools such as cell-penetrating peptides, aptamers, or other
high molecular weight ligands which might interfere with RISC entry. Indeed, pilot
in vivo studies discussed above in Sect. 2.3 demonstrated the successful use of
DsiRNAs covalently conjugated with both large aptamer and CpG-motif oligonu-
cleotides to facilitate delivery.
Synthesis of siRNA and DsiRNA duplexes is available from a variety of com-
mercial sources from very small scale to multi-gram scales to support all research
and preclinical needs. A smaller number of suppliers are certified to produce the
cGMP quality synthetic oligonucleotides needed for pharmaceutical use in humans.
Currently, most cGMP manufacturers employ commercially available synthesis
platforms, such as the GE Healthcare OligoPilot™ and OligoProcess™ synthesiz-
ers, or the Asahi Kasei TechniKrom ® platform, which are capable of doing synthe-
ses in millimole to mole scales, resulting in greater than kilogram yields of final
product. Methods to produce cGMP quality synthetic oligonucleotides are well
established, largely thanks to the many years of experience gained from clinical tri-
als done using single-stranded antisense oligonucleotides [ 106, 107 ] .
Acknowledgments The authors thank Kim Lennox for critical reading of the manuscript and
Dr. Joe Dobosy for assistance with the figures. We further thank Dr. John Rossi, Dr. Dongho Kim,
and all members of the research laboratories at Integrated DNA Technologies for their contribu-
tions towards the development of the Dicer-substrate siRNA technology.
Con fl ict of Interest Statement MAB and SDR are employed by Integrated DNA Technologies
(IDT), which sells compounds similar to those described herein. IDT is, however, not a publicly
traded company, and the authors do not hold any stock or equity in IDT. MAB is a scientific
cofounder of Dicerna Pharmaceuticals and is a member of their Scientific Advisory Board.