RNA interference (RNAi) is an ancient genetic regulatory mechanism that modern-day scientists are trying to harness. Since its discovery more than 10 years ago, RNAi R&D has run the gamut from excitement to disappointment to cautious optimism. Despite the roadblocks and difficulties, RNAi remains a powerful tool not only for basic science but also as a potential therapeutic.
RNAi is a natural process for gene silencing mediated by RNA silencing machinery and facilitated by small RNAs that bind to and degrade messenger RNAs (mRNAs). Part of its promise stems from its exquisite specificity to seek out and destroy its target without affecting regulation of other genes. GTCbio’s recent “RNAi Research & Therapeutics” conference showcased new advances in the field including novel chemistries for synthetic silencing oligonucleotides and highlighted emerging approaches for RNAi-based therapies.
In the past decade, microRNAs (miRNAs) have emerged as a new class of regulatory molecules that modulate gene expression. Operating at the post-transcriptional level, the small noncoding RNAs impact a diverse array of processes ranging from development to apoptosis.
Although antisense technology has been used for more than 30 years as a means to reduce the expression of specific RNAs, introduction of synthetic anti-miRNA oligonucleotides (AMOs) represents a new application of antisense methods.
“AMOs are typically 24 bases or less, and are usually made as full-length reverse complements to the mature miRNA,” noted Kim A. Lennox, senior research assistant at Integrated DNA Technologies (IDT; www.idtdna.com). “They can also employ chemical modifications both at the internucleoside linkages as well as the 2´ hydroxyl position of the ribose sugar (i.e., 2´-O-methyl RNA). Various modification patterns can have a large impact on the potency, specificity, and toxicity profiles of AMOs. Increased binding affinity (i.e., high Tm) usually correlates with increased potency but comes at a price of decreased specificity.
“Finding the right balance between potency and specificity can be challenging. Some modifications can also cause toxicity, so working with nontoxic compounds is important, especially if in vivo use is planned.”
Lennox said that, in addition to low toxicity, other desirable features for an AMO include nuclease resistance, increased binding affinity, and high specificity. “While AMOs synthesized using the 2´-O-methyl chemistry have several advantages, such as increased Tm when duplexed with miRNA targets and resistance from degradation by endonucleases, further improvements in stability and potency are needed for these reagents to reach their full potential.”
IDT recently developed a non-nucleotide modification called ZEN. According to Scott D. Rose, Ph.D., director of molecular biology, “ZEN dramatically improves the performance of 2´-O-methyl AMOs when inserted into the sequence near both ends. The ZEN modification is a non-nucleotide napthyl-based compound. This new AMO design has many advantages, including: 1) protection from exonucleases, 2) enhancing Tm of the oligonucleotide without sacrificing specificity compared to other Tm enhancing modifications, and 3) low toxicity.”
Dr. Rose indicated that a recently published study by Eran Hornstein’s laboratory at the Weizmann Institute illustrated the utility of IDT’s new ZEN-AMO chemistry by using these compounds to suppress specific miRNAs in pancreatic islet cells, demonstrating a role for these miRNAs in insulin regulation.
The ZEN chemistry is currently available for custom-ordered oligos and the new ZEN-AMO product line will appear in their catalog in the first quarter of 2012.
Dual 3' Olgo Ends
Nicola La Monica, Ph.D., vp of biology at Idera Pharmaceuticals, reported on the company’s gene-silencing oligonucleotides, termed GSOs. “Our objective for developing these was to identify novel oligonucleotides to modulate gene expression that decreased the common problems associated with RNAi such as delivery and lack of potency.”
GSOs are single-stranded oligonucleotides with two exposed 3´-ends whose 5´-ends are joined via a linker. “The 5´-ends of oligos are known to be responsible for immune activation through interaction with the toll-like receptor 9 (TLR9), while the 3´-ends are required for gene-silencing activity. The GSO design accomplishes two things. By blocking the 5´-ends, immune activation is reduced while the 3´-ends allow for target mRNA processing. Since the GSOs can be delivered systemically, there is no complication of needing a specific delivery technology.”
To validate the technology, company scientists synthesized RNA and DNA GSOs targeted to vascular endothelial growth factor (VEGF), the adapter protein MyD88 and toll-like receptor 9 (TLR9). “The GSOs varied in length (15–25 mers) with each linking two identical oligonucleotides complementary to the target mRNA. In comparing the GSOs to traditional antisense compounds, we observed that 19 mers of RNA and DNA GSOs had the highest potency for gene silencing in vitro and in vivo.”
The company will continue to improve on the technology and expand the list of targets that can be knocked down. “We are also working to better understand the mechanism of action. Because our studies demonstrated that the GSOs complementary to various mRNA targets showed similar results, we feel the technology will have broad applicability.”