If there is one thing that researchers agree on, it’s that now is a good time to be working with RNA interference (RNAi). RNAi is a natural cellular mechanism that regulates gene expression at the stage of translation by degrading the mRNA or blocking translation. It can also alter the level of transcription of specific genes.
Double-stranded RNA (dsRNA) triggers a series of biochemical events that culminates in sequence-specific suppression of gene expression. “Long dsRNAs have been employed for many years as a means to modulate gene expression in plants, yeast, and C. elegans,” noted Mark Behlke, M.D., Ph.D., svp of molecular genetics and CSO at Integrated DNA Technologies (IDT; www.idtdna.com).
“Similar attempts in higher organisms failed due to interferon activation, however we now know that short RNA duplexes can be safely used in mammalian systems both in vitro and in vivo. The technology has rapidly matured, thanks in large part to all that was learned over the past 20 years using antisense oligonucleotides. RNAi is now routinely employed in vivo as an experimental tool and numerous groups are vigorously pursing the use of RNAi compounds as therapeutics. Several siRNA drugs are already in clinical trials and more are in preclinical development.”
The RNAi pathway is initiated by the enzyme dicer, which cleaves long dsRNA molecules into short fragments of 20–25 base pairs. One of the two strands of each fragment, known as the guide strand, is then incorporated into the RNA-induced silencing complex (RISC) and paired with complementary sequences.
The most well-studied outcome of this recognition event is post-transcriptional gene silencing. This occurs when the guide strand specifically pairs with an mRNA molecule and induces the degradation by argonaute, the catalytic component of the RISC complex. Another outcome is epigenetic changes to a gene—histone modification and DNA methylation—affecting the degree to which the gene is transcribed.
RNAi targets include RNA from viruses. These targets also play a role in regulating development and genome maintenance. Breakthroughs in this technology are making it possible to access biological information that was not possible until recently.
The selective and robust effect of RNAi on gene expression makes it a valuable research tool, both in cell culture and in living organisms, because synthetic dsRNA introduced into cells can induce suppression of specific genes of interest. RNAi may also be used for large-scale screens that systematically shut down each gene in the cell, which can help identify the components necessary for a particular cellular process or an event such as cell division. Exploitation of the pathway is also a promising tool in biotechnology and medicine.
Discussion of these new technologies took center stage at the “Second Annual RNAi World Congress” in Boston last month, where presenters described some of the new opportunities that lay ahead for research and drug discovery. “This is a really exciting time to be in the field,” said Dr. Behlke.
Design and effective delivery of synthetic RNAi compounds are essential for therapeutic applications, explained Dmitry Samarsky, Ph.D., vp of technology development for RXi Pharmaceuticals (www.rxipharma.com), whose offerings include rxRNA compounds. “rxRNA is a next-generation product that can be up to 100 times more potent than conventional siRNAs.”
“The shortcoming with original unmodified siRNA is nuclease instability. The modifications we have made demonstrate nuclease resistance and are potentially more specific for their intended target. The other thing is that we can use compounds in the modification to block interferon. These three pieces are the foundation to this technology.”
RXi’s founding scientists recognized early that the key to therapeutic success with RNAi lies in delivering intact RNAi compounds to the target tissue and the interior of the target cells, noted Tod Woolf, Ph.D., CEO. “RXi will work with chemically synthesized RNAi compounds that are optimized for stability and efficacy. We intend to rely on a combination of delivery at the site of action and formulation with delivery agents to achieve optimal delivery to specific target tissues.”
Dr. Samarsky also discussed RXi’s nanotransporters, which have been used to deliver RNAi compounds to the mouse liver and obtain exceptionally low dose (1 mg/kg) gene-specific inhibition, the company reported. Delivery to the liver is critical for many metabolic targets, including diabetes and obesity. In addition, nanotransporters are of a defined size and are readily formulated.
FANA-Modified Nucleic Acids
“Topigen is focused on applying RNA approaches to respiratory diseases and finding innovative approaches to the discovery of new and better treatments for these diseases,” said Nicolay Ferrari, Ph.D., director of pharmacology at Topigen Pharmaceuticals (www.topigen.com).
The company’s focus is on therapy for inflammatory respiratory diseases such as chronic obstructive pulmonary disease (COPD) and asthma. Its core technology is based on a proprietary chemistry and a multitarget approach using antisense oligonucleotides. The company’s products are designed to be inhaled to act locally and directly on lung tissue and thereby increase potency while reducing potential side effects by minimizing systemic exposure.
Topigen’s technical strategy is to downregulate multiple and overlapping pathways in inflammation. The effectiveness of this approach, which circumvents the redundant nature of the human asthma/allergy inflammation response, has been proven in human trials of Topigen’s lead compound TPI ASM8 for asthma, according to the company. In the case of COPD, Topigen’s new antisense-based therapeutic, TPI 1100, targets two isoforms of the phosphodiesterase enzyme (PDE4 and PDE7) and is being developed to reduce lung inflammation and halt the progression of disease.
Dr. Ferrari focused on the company’s RNA-targeting technology and the use of its 2´-deoxy-2´-fluroarabinucleic acid (FANA™)-platform, which originated from McGill University and was licensed exclusively to Topigen, said Dr. Ferrari. “It’s a chemical modification of natural DNA. What differentiates FANA from other chemical modifications is that it promotes RNaseH activity,” he added, “which is important for optimal efficacy of an antisense-based therapeutic.”
Natural DNA is labile and not very resistant. The initial goal of chemical modification is to increase stability as well as the activity of the oligonucleotide. “By increasing potency and stability, we can reduce the dose—it’s true that more is not necessarily better in dose ranging,” explained Dr. Ferrari.
“The key advantage of FANA is the flexibility it allows us when designing oligonucleotides to increase potency and duration of action. When combined with our delivery by inhalation, it limits systemic exposure, which is essential in limiting side effects associated with systemic delivery of oligonucleotides.” FANA represents a new chemistry that can improve properties of RNA-targeting agents including antisense, siRNA, and aptamers, Dr. Ferrari concluded.