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Ramping Up Small RNAs

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Researchers could debate the semantics of whether we’re truly in a microRNA revolution, but there’s no debating the fact that these small RNAs have at last come into their own. A quick PubMed search comes up with almost 1,000 hits in 2007, twice that of the preceding year.

In fact, the more tools that are created to study these RNAs, the more scientists discover about how they work and what they do. Researchers steeped in the field say that much of the current research revolves around gene expression profiling studies. After isolating the miRNAs of interest, functional assays can be run to up- or down-regulate these miRNA genes in order to find out what role they play in both normal cellular processes and disease pathology.

Yale’s Frank Slack, a pioneer in miRNA research, looks at how miRNAs are involved in development and how they control diseases — particularly lung, breast, and brain cancer. “We want to try and understand how some of the known miRNAs, in particular the ones that are conserved across many different animal species, actually control the various aspects of development,” Slack says. In addition, they look for new roles as well as new miRNAs that haven’t previously been found to play a role in these processes.

With the technology becoming more user-friendly and reliable, Slack says that most people are doing gene expression studies — likely for the inherent value in an miRNA expression profile. “First of all, we can use it as a diagnostic because that profile is specific for that disease,” he says. “Secondly, the miRNAs that are most abundantly changed, either up or down, in the disease state versus the normal state, are often miRNAs that are involved in the disease state itself.”

With the latest advances in small RNAs, Slack thinks the field is headed for a “major explosion,” he says. “We’re missing half of the fun by just focusing on the protein-coding genes. We’re going to learn that these small RNAs are probably playing as important a role in our bodies and our development as maybe all the protein-coding genes and transcription factors.”

For the genetic screening work, Slack says, his team performs bioinformatic screens in C. elegans to find potentially useful miRNA targets, following that up with RNA isolation kits to identify them. Moving on to mouse models, he and his colleagues use transgenics to both over- and underexpress miRNAs to see whether they can “find miRNAs that are associated with those diseases — and can we use miRNAs to perhaps alter the course of those diseases?”

Expression and beyond

Meanwhile, other researchers are using similar tools for both gene expression and pathway elucidation research. Jens Kurreck, a biochemist at the Free University Berlin, splits his time between using RNAi for target validation in the neuropathic pain pathway and for viral knockdown in the search for an RNAi therapeutic for viruses. In the world of miRNA, Kurreck also has some gene expression analysis underway. In studying the cellular changes that happen upon infecting cells with the Coxsackievirus, Kurreck first looks at the miRNA expression pattern using chips to “see that certain miRNAs are differentially regulated after infection with the virus,” he says. After picking several important miRNAs, he does follow-up functional assays, selectively up- or down-regulating the miRNA to see what happens.

Common ways to inhibit miRNA expression, Kurreck says, are to use various forms of antisense molecules, including locked nucleic acids and antagomirs for in vivo knockdown; these are specially designed, single-stranded nucleic acid molecules that bind to the RNA and inhibit its function. In the past, antisense was commonly used for gene knockdown, but now that RNAi has proven more effective for this purpose, antisense has found an additional and surprisingly effective home in the area of miRNA inhibition.

Josh Mendell, an assistant professor of pediatrics, molecular biology, and genetics at Johns Hopkins University, also uses these advancing tools of the trade to elucidate function in cancer tumorigenesis pathways. He and his team have recently shown that Myc, a transcription factor that plays a large role in cancer, up-regulates a group of miRNAs, the miR-17-92 cluster, while down-regulating about 10 to 20 others. “What we’ve shown is that one of the important things that happens when Myc becomes hyperactive is that it really changes the program of miRNAs that are expressed in a cell, and those changes are really important in promoting abnormal proliferation and tumorigenesis,” Mendell says.

In studying an miRNA of interest, Mendell says, his group tries to both enforce and inhibit expression. “Those are complementary approaches, and then we examine the phenotypes that result from those manipulations,” he says. They clone retroviruses and then express them into a gene of interest to overexpress an miRNA; to inhibit one, they take the same knockout approach as Kurreck — injecting antisense oligos directly into cells for transient inhibition. “That’s the most common way, although there’s been a lot of interest in generating methods to stably inhibit a microRNA,” he says.

The vendors’ view

Much of this work has been made possible with the new miRNA tools on the market. While vendors continue to improve tools for RNAi research, miRNAs are also getting a lot of attention. As they learn more about how this endogenous silencing mechanism works inside cells, vendors continue to develop tool sets for miRNA research.

According to Peter Welch, director of Invitrogen’s gene expression profiling research and development, expression analysis is one area that is growing rapidly. “You just break open a cell and look to see what endogenous miRNAs are being expressed, because that pattern ultimately dictates the fate of the cell or how it’s going to respond to its environment or other various factors,” he says. To that end, Invitrogen launched a product called NCode, an miRNA analysis system built off miRBase, the Sanger miRNA sequence database. Invitrogen’s latest release of the miRNA expression array will have about 1,500 unique miRNA features, including all of the Sanger content (about 1,100 human miRNAs) as well as about 460 novel miRNAs that were developed or discovered by deep sequencing, according to Welch. “So this is really the first example of a commercial product where people have taken the data from a deep sequencing reaction and put it onto an array and made it available,” he says.

Applied Biosystems, which acquired RNAi reagent leader Ambion a few years ago, also has a full range of products for miRNA research, including those for sample preparation and RNA isolation, detection, and quantification (for gene expression profiling assays), and pre-miRs and anti-miRs (for functional research and pathway elucidation). “The field’s really exploded on all fronts,” says Criss Walworth, director of gene expression assays at ABI. While her company has a lot of customers who are interested in profiling miRNA content from a variety of different tissues and cells,  she says that functional analysis — using tools like pre-miRs as miRNA mimics and anti-miRs as miRNA inhibitors in order to see what different miRNAs are doing in the cell — has also become of great interest to miRNA researchers. Walworth says she thought the field would quickly elucidate function and move on to using miRNAs for biomarkers and therapeutics, but “it hasn’t played out that way at all.”

Sidebar:
Cequent’s Approach: Put Bacteria to Work

Cequent Pharmaceuticals focuses on developing RNAi for therapeutics. To bypass mistargeting of tissues or poor cellular uptake, Cequent engineered bacteria that can deliver siRNAs to cells of interest; the bacteria are then degraded and passed out of the cell.

“Most people are using synthetic siRNAs and trying to find ways to get them into cells, whereas we don’t synthesize RNA at all. We actually program bacteria to make a precursor and get inside the cell and deliver it there,” says Peter Parker, president and CEO. “RNAi works all the time, if you get it there.”

The bacteria, says research VP Johannes Fruehauf, are not harmful, even though they “have scary names, like E. coli or Salmonella.” With the focus on creating better, deliverable therapeutics for several diseases, critics have asked whether ingesting live bacteria will be more harmful than helpful to patients. Fruehauf thinks not. “[These] are engineered bacteria that have very defined genomic properties into which we build nutritional deficiencies and other safety measures to prevent them from ever being able to cause an infection, or to even colonize.”

Sidebar:
The Scoop on siRNAs: More Tweaked, More Stable

MicroRNAs aren’t the only small RNAs getting development attention. A number of vendors continue to optimize siRNAs through different approaches, with the main goal of developing a deliverable therapeutic. Most of the common chemical and structural modifications used today change the chemical structure of the backbone of the RNA molecule.

While chemical and structural modifications to siRNA molecules have already improved silencing, they still need tweaking. Iowa-based Integrated DNA Technologies is the first company to offer Dicer-substrate siRNAs, based on its collaborative work with City of Hope’s John Rossi, among others. Boston-based Dicerna Pharmaceuticals plans to begin marketing therapeutics based on this technology in the near future.

IDT and Rossi co-authored a 2005 paper that found a novel way to decrease off-target effects using what is now known as Dicer-substrate siRNA. At 27 oligomers, their Dicer substrate is longer than the typical 21-mer siRNA; it makes the molecule look more similar to an endogenous double-stranded RNA. Overall it results in more specific, more potent knockdown. “All we did was accidentally stumble on the fact that if you make these RNAs longer, so that they now become substrates for Dicer, that they can actually more potently incorporate into RISC and give more potent knockdown,” Rossi says. “Primarily the 27-mer is what we’re staying with. We can go up longer, but we also want to make sure that we don’t trigger any interferon responses,” Rossi says.

The main goal of most chemical modifications is to make the siRNAs more stable. “By adding the stability you sort of make these molecules more amenable to an eventual therapeutic, which is where a lot of people are ultimately heading toward,” says Invitrogen’s Peter Welch. Invitrogen recently upgraded its Stealth product line to Stealth Select, which utilizes both chemical modifications and additional bioinformatic filters to create molecules that have fewer off-target binding effects. “What we found is if you place the chemical modifications, like Stealth modifications, on these, the cell doesn’t really see it as a double-stranded RNA, and so you evade that interferon response while still getting all the benefits,” he says.

Meantime, with the recent release of its Silencer Select siRNA product line, ABI’s Ambion has taken its RNAi product line “to the next level,” according to David Dorris, senior director and general manager for cell biology and RNAi at ABI. In an effort to improve knockdown efficiency, the Select products aim for better design and improved potency. To improve specificity, ABI has incorporated locked nucleic acids into the siRNAs as well as several bioinformatic filters — including one that can identify motifs responsible for toxicity. “The design algorithm by itself is not good enough,” Dorris says, “[and] the bioinformatic filters by themselves are certainly not good enough.” One challenge to developing robust products is that “they’re gene-specific products, and the gene specificity is only as good as the data that’s in the public realm,” he adds.

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