by Catherine Shaffer
RNAi technology has advanced far beyond the speculative phase, and researchers are looking for higher quality siRNA in greater quantities and wider gene coverage than ever before. Much of the spike in demand comes from RNA interference drug screens for drug target identification or validation. Large-scale screens often follow successful small-scale studies, and can consume upwards of 500 mg of siRNA in the subsequent validation studies—some organizations will even order a gram at a time.
Although this is an exciting advance in the field, there are a couple of pitfalls that come with the territory, including off-target effects, difficult-to-transfect cell lines, identifying a strong phenotypic effect, quality control, and GMP compliant products.
Off-target effects are probably the most frequent bugaboo of siRNA technology. If the siRNA oligo has sequence homology to non-targeted mRNA, it can wreak havoc with the RNA interference experiment. One strategy for avoiding off-target effects comes from Sigma-Aldrich. Their MISSION esiRNA line of endonuclease-digested siRNAs mirrors their extensive library of standard siRNA. The esiRNAs are prepared by first amplifying a region of the gene with the highest number of effective siRNAs. This region is then enzymatically digested to form a pool of hundreds of different siRNAs. Says Steve Suchyta, market segment manager, functional genomics at Sigma Research Biotech, “These multiple silencing triggers have been shown to give very effective knockdown with very low off-target effects, and in some cases far fewer off-target effects than siRNA.” MISSION esiRNA is available as individual, custom libraries, and as whole genome libraries, and come with a guarantee of at least 70% knockdown at the mRNA level.
Another issue with RNAi screens is the ability to detect the phenotype in the assay. Having a strong phenotype is crucial when determining whether a gene is a "hit" or not. “With MISSION esiRNA, essentially a large pool of siRNA, the knockdown effects are reinforced, producing strong phenotypes, even in some cases where individual siRNA themselves were not effective although the siRNA are knocking down the target mRNA,” says Suchyta.
Digested siRNA products can be made using endonuclease, or with the “biologically correct” enzyme Dicer. This is the strategy employed by Integrated DNA Technologies (IDT) in its DsiRNA product line. DsiRNAs are processed by Dicer and are 27-mer length; standard 21-mer siRNAs mimic Dicer products and avoid Dicer processing. Users have noted increased potency and efficacy in gene silencing with this type of oligonucleotide. The effect is most dramatic at low doses. Says Mark Behlke, CSO of IDT, “When using high doses of siRNA you often won't see differences between these and 21mers. If you're using low dose, you can minimize side effects, and we think there's a real potency benefit.”
IDT has noted an increased demand in terms of quantity and quality of siRNA products. “People have success with small scale studies, and are going into animal studies...that's the kind of exciting trend that we're seeing in the past couple of years, compared to 4-5 years ago. It's just what you'd hope, people having success and moving this forward, going from cell culture to animal work.” RNA interference studies in live animals require a higher degree of quality control, and IDT sells “in vivo grade” siRNA up to 10 gm scale. For a product that may eventually be used in human studies, that is yet another jump up in quality, requiring a GMP compliant supplier. IDT does not provide GMP oligonucleotides for human clinical trials. They partner with Agilent Technologies to help their customers make this transition. But according to Behlke, it's never too early to start thinking about your clinical supply: “We strongly encourage people to begin work with their GMP provider earlier rather than later. Once you're fully into rodent tox studies, you're better off doing those with your GMP material.” Using a consistently high quality product from the research stage to the clinical stage can make this transition less painful.
Not all siRNA products are equal. Endonuclease or Dicer digestion can greatly improve the sensitivity and potency of RNA interference, depending on the application. Large libraries of siRNAs are available for most of the model systems used in today's research. Many vendors now have the ability to manufacture siRNA in gram quantities, and will work closely with customers and GMP suppliers to ensure a consistent product from early research to late-stage clinical development.