June 1, 2008 (Vol. 28, No. 11)

Technology’s Viability in Drug Development Is Finally Established

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.

Therapeutic Applications

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.

Gastrointestinal Targets

Another novel technology debuting at the congress was Transkingdom RNAi, (tkRNAi), which delivers therapeutic RNAi to the gastrointestinal tract. Johannes Fruehauf, M.D., vp, research, at Cequent Pharmaceuticals (www.cequentpharma.com), noted that the approach had its genesis in research from the Beth Israel Deaconess Medical Center/Harvard Medical School. “We had an interest in doing RNAi research and applying it to colon cancer and other GI diseases. We really worked on this for about four or five years at Beth Israel before coming over to Cequent,” said Dr. Fruehauf.

The company’s first products, now in preclinical development, target colon cancer. The tkRNAi technology takes a Trojan horse approach to solve one of the key challenges to RNAi therapeutics—delivery of the RNAi into the cell. Cequent uses live attenuated bacteria to produce and deliver RNAi from the luminal side to the GI mucosa, allowing oral administration.

“Cequent designed its powerful tkRNAi technology to deactivate specific disease-causing genes safely and effectively using nonpathogenic bacteria as an engine to produce and deliver RNAi directly into cells,” Dr. Fruehauf noted.

In addition to findings with oral application, Dr. Fruehauf’s group found that, upon intravenous administration, it was possible to obstruct certain tumors in human xenografts in mice. Currently, his group is conducting a monkey toxicology study. “These results provide an example of trans-kingdom RNAi in higher organisms and suggest the potential of bacteria-mediated RNAi for functional genomics, therapeutic target validation, and development of clinically compatible RNAi-based therapies,” said Dr. Fruehauf.

There are other active programs at Cequent as well. “We are also researching ways to treat inflammatory bowel disease,” reported Dr. Fruehauf. “The approach we take will be similar to the one we are taking with cancer and polyps in the GI, but we are looking into a range of other disease targets related to inflammation and the regulation of inflammation in the gut. Hopefully, we will be able to tilt the balance toward improving inflammatory diseases. A third program is investigating the use of tkRNAi against HPV and cervical cancer. This tool is exquisitely useful in targeting epithelial cells, and there is a story to tell.”

Another technology for improving RNAi applications making its debut hails from RiboTask (www.ribotask.com). “Unlocked nucleic acid (UNA) is an acyclic form of RNA that is remarkably useful as a novel constituent of siRNA duplexes,” said Jesper Wengel, Ph.D., cofounder and chairman. “It’s a molecule that’s easy to make and can be produced cheaply.”

UNA can be used in RNAi and siRNA constructs, potentiating other chemical modifications and allowing molecules to be further stabilized. “It unlocks the use of chemically modified siRNAs, which can reduce off-target effects, induces high biostability, and allows antisense strand modification.” Dr. Wengel presented results from cellular studies that show siUNA constructs display improved potency, reduce off-target effects, and increase biostability relative to unmodified siRNA duplexes.

Thermo Fisher Scientific (www.thermo.com/dharmacon) has developed a molecule for lipid-independent delivery in a wide variety of cell types. The presentation by Kirk Brown, Ph.D., field application scientist, was geared around Dharmacon Accell siRNA.

Dharmacon Accell siRNA reagents are modified for uptake without a separate delivery reagent. “Because they do not require transfection reagents, Accell siRNA reagents provide silencing of the target gene without the lipid toxicity and off-target effects that cause misleading phenotypes,” explained Dr. Brown. “And they have been found to effectively silence target genes in cell types that are typically difficult to transfect, e.g., neuronal and suspension cells using standard delivery methods.”

Michael Deines, global director of marketing, genomic technologies, Thermo Fisher Scientific, noted that there were other considerations that went into the development of Accell. “We wanted to be able to allow RNAi experiments in biologically relevant cell types, which is crucial to research efforts,” said Deines. “But also the delivery technology is key; it’s such that it allows repeated application of Accell siRNA to provide extended duration gene knockdown with only minimal effects on cell viability and the innate immune response. These attributes will greatly broaden the range of biological questions and cell types that can be investigated by researchers using RNAi.”

“This new technology,” Dr. Brown added, “has given us success in cell types that have been hard to reach and access in the past. Keep in mind, however, that it’s the newest development. It doesn’t replace existing technologies but it’s enabling technology that makes more cell types accessible to RNAi.”

miRNA microarray expression profiling depends on robust labeling of the RNA sample, noted Shannon Bruse, senior scientist with Mirus Bio (www.mirusbio.com). “We’re looking at one component in the process, but it’s one that has importance,” said Bruse. “What we are seeing is that there are subsets of microRNAs that can’t be labeled or sufficiently labeled—one class can’t be labeled at all and the other one is labeled inefficiently.”

Use of Dicer Substrate siRNAs

Dicer-substrate siRNAs (DsiRNAs) have recently been employed for in vivo studies using intraperitoneal and intrathecal routes of administration. “IDT got into RNAi research in collaboration with John Rossi at The City of Hope and the Beckman Research Institute five years ago,” explained Dr. Behlke. In vivo, long dsRNAs are cleaved by the RNase III class endoribonuclease dicer into 21–23 base duplexes having 2-base 3´-overhangs. These species, called small interfering RNAs (siRNAs), enter the RISC and serve as a sequence-specific guide to target degradation of complementary mRNA species.

Typically, siRNAs are chemically synthesized as 21 mers with a central 19 bp duplex region and symmetric 2-base 3´-overhangs on the termini, reported Dr. Behlke. These duplexes are transfected into cells lines, directly mimicking the products made by dicer in vivo. Most siRNA sequences can be administered to cultured cells or to animals without eliciting an interferon response.

“We observed,” added Dr. Behlke, “that the use of slightly longer sequences that were substrates for dicer showed improved potency, which we theorize relates to participation of dicer in RISC loading. We are now focusing on the use of these compounds in vivo.”

IDT recently completed a collaborative study with the laboratory of professor Phillipe Sarret at the Université de Sherbrooke in Quebec. The collaboration studied the use of DsiRNA to knockdown the GPCR NTS2 (neurotensin type 2 receptor) in rat spinal cord and dorsal root ganglia. The RNA duplexes were administered by intrathecal injection in a cationic lipid slurry. Stimulation of NTS2 with a chemical agonist resulted in analgesia. Pain responses were monitored in treated animals by dipping their tails in hot water with and without the chemical agonist.

“The anti-NTS2 DsiRNA treated animals showed a marked difference of response to the test stimulus,” said Dr. Behlke. “We recorded differences of up to five seconds, which is quite a long time for a rat to sit with its tail in hot water. While interesting, this work mainly represents a pilot study to demonstrate the feasibility of using DsiRNA to study pain pathways in rats. We were amazed at the low dose it takes to get knockdown—we used 1 mcg/200 g rat, which is only a 0.005 mg/kg dose.” Modulating CNS disease and affecting brain processes is clearly possible, but better methods of delivery are going to be needed to move this approach from a research tool into the clinic, noted Dr. Behlke.

miRNA Labeling Methods

Bruse’s group recently embarked on a study to compare and contrast the effects of enzymatic labeling and chemical labeling. Their discovery, which was presented at the meeting, was that the labeling issue is significant. “The subset we are talking about is 5–20% of all experiments, which means that labeling causes a significant amount of bias in the results,” said Bruse. “Enzymatic labeling systematically misses specific microRNAs, whereas chemical labeling results in unbiased detection of all microRNAs.”

“We have some of the figures up on the website showing the results of our findings,” said Bruse, adding that there haven’t been any papers published on the subject of labeling and the possible biases different labeling methodologies might impose. “This is cutting-edge stuff,” he noted. “The story is in the labeling method and helping scientists understand how this choice impacts the data.”

“What you can do with RNAi technology is truly amazing,” Dr. Behlke concluded. “These tools will help researchers solve bigger questions and help develop treatments for metabolic diseases, viral diseases, and more.”

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