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Feature Articles : Nov 1, 2011 (Vol. 31, No. 19)

Cutting-Edge Approaches and Applications for RNAi

Powerful Tool's Exquisite Specificity Fuels Its Utility Across a Broad Range of Research Channels
  • Kathy Liszewski

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.”

Reconstructing Bases

Most approaches to improve on short interfering RNAs (siRNAs) have focused on the ribose moiety of nucleotides. A largely unexplored but potentially significant route to modulate siRNA properties is the base itself, suggested Peter A. Beal, Ph.D., professor of chemistry, University of California-Davis.

“Modifying nucleobases within a sequence can exert profound effects on the chemical, biological, and physical properties and interactions of oligonucleotides. Immune stimulation mediated by RNA oligonucleotides harboring certain sequence motifs, such as GU-rich regions, is a significant hurdle to the development of safe and effective RNAi therapeutics.

“We addressed this challenge in a series of studies in which we employed novel nucleobases in an oligonucleotide mimic of microRNA-122 (miR-122). Because it is already known that modification of the ribose 2´-position can reduce immunostimulation, we constructed our study to also include these modifications for comparison.”

Dr. Beal and colleagues utilized analogues of adenosine and guanosine that contained cyclopentyl and propyl minor-groove projections. “siRNAs have an A-form helix structure that contains both a major groove and so-called minor groove. The latter is the site of binding for many RNA-interacting proteins. We synthesized guanosine analogs with base modifications that affected the minor groove. Further, we substituted these analogs at different positions within reported immunostimulatory motifs. To test for RNAi activity, we measured the knockdown of two native miR-122 targets (Necap2 and Slc7a1).”

The results were unanticipated. “To our surprise, we found single nucleotide positions throughout the guide strand of an miRNA-122 mimic acted as immunostimulatory hot spots of activity that did not correspond with putative motifs (e.g., GU-rich regions). We believe these hot spots are locations where RNA and Toll-like receptors interact. Thus, modification of nucleobases at specific positions can control immunostimulatory properties of siRNAs in ways not predicted with traditional immunostimulatory motifs.”

Promoter Impact

Successful RNAi-mediated knock-down requires efficient delivery of the payload. Traditional methods (e.g., lipid-based reagents) may not work well for certain cell types such as neurons and hematopoietic cells. Viral vectors offer an alternative system for delivery.

“Viral biology can be engineered to deliver genetic material into cells very effectively and modulate gene expression for both short- and long-term functional studies,” explained Devin Leake, Ph.D., global director of R&D genomics, Thermo Fisher Scientific.

According to Dr. Leake, a viral vector system needs to address three key parameters. “It must be able to effectively deliver genomic content, have a very broad tropism, and allow for the long-term expression of that content. This requires the careful design of the short hairpin RNA (shRNA), the vector backbone, and choosing the best promoter for the particular cell type utilized.”

In creating the Thermo Scientific SMARTvector2.0® Lentiviral shRNA platform, the company designed a microRNA-adapted expression scaffold to maximize accurate processing of the silencing sequence while minimizing the potential for off-target effects.

“We rigorously tested a panel of miRNAs and selected one that could consistently and efficiently be processed by the endogenous RNAi-silencing machinery. It is also necessary to have rationally designed highly functional gene targeting sequences. The SMARTvector2.0 algorithm selects optimal shRNA targeting sequences and includes seed-based filters to reduce toxicity.”

Another important feature of a lentiviral vector system is the promoter. Although the company initially included the promoter from cytomegalovirus (CMV), it will soon be releasing an a-la-carte series of promoters that investigators may choose.

“The CMV promoter may be too weak in some cell types. There is no universally optimal promoter, so we have developed a series of SMARTvector backbones with the option of seven different promoters, called the Thermo Scientific SMARTchoice system, which will provide researchers with greater flexibility and more choices for successful RNAi experiments.”

Thermo Fisher Scientific custom-builds the lentiviral system using the requested promoter and the appropriate sequence for the client’s target of interest. It plans to release the system in the first quarter of 2012.

RSV miRNA Tactics

Respiratory syncytial virus (RSV) infects many children by the age of 2 and causes >100,000 hospitalizations each year in the U.S. It also strikes the elderly resulting in more than 14,000 deaths. Currently there is no safe and effective vaccine against RSV. Further, antiviral drugs are limited. Clinical trials are currently being conducted using RNAi therapeutically against RSV. Additionally use of a humanized mAb (palivizumab) against another RSV protein provides an option for treating infants at risk.

Ralph A. Tripp, Ph.D., Georgia Research Alliance chair and professor of infectious diseases, is investigating how RSV modulates human miRNAs. “Studies are showing that many viruses like RSV dysregulate host miRNAs to alter host defenses, and that this process can be attributed to specific virally encoded proteins that assist infectious processes. Because single miRNAs are predicted to regulate multiple genes and affect hundreds of processes, a goal of my lab is to determine how RSV infection influences global gene expression in respiratory epithelial cells.”

Dr. Tripp’s group infected cultured cells with RSV and RSV mutant viruses deficient in single genes and utilized miRNA reporter assays to assess cellular dysfunctions. Next they employed computational algorithms to predict miRNA target genes and found and validated sets of genes targeted by the miRNAs.

“Our studies demonstrated that in epithelial cells, RSV infection induced five specific miRNAs and repressed two. The RSV-specific proteins modulated miRNAs that affected cell cycle and chemokine genes, and suppressed cytokine signaling genes that control antiviral cytokine responses.”

These findings may translate into improved therapeutics eventually. “We’ve spent over 35 years trying to produce a vaccine against RSV. Now that we better understand how it controls host immune responses, we have the opportunity to create a new vaccine using miRNAs to create a more robust immune response to RSV. Additionally, development of miRNA agonists or inhibitors could also be envisioned for inhibiting an active RSV infection. Understanding the mechanisms used by RSV to control the host’s response to infection will allow us to develop improved intervention strategies that are safe and effective.”