Genomics & Proteomics

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Making Sense of RNA Interference Methods

Four years after its discovery, RNAi has changed how drug discovery scientists view genes and proteins

click the image to enlarge

siRNA may be created synthetically and inserted into the cell (arrow 3), or obtained by processing of dsRNA (arrows 1, 2). One of the strands of the siRNA duplex targets a homologous (complementary) region in a specific gene mRNA (arrow 4). The mRNA is then cleaved (arrow 5), and the resulting pieces are degraded (arrow 6), effectively silencing the gene. (Source: Compugen)
Since the early days of molecular biology, ribonucleic acid (RNA) has taken a back seat to deoxyribonucleic acid (DNA). DNA, after all, holds the “instructions,” whereas RNA merely carries them out. RNA’s status was upgraded in the early 1990s when professor Victor Ambros at Darmouth College found RNA strands in Caenorhabditis elegans that interfered with the worm’s messenger RNA (mRNA). In 1999, when professor Andrew Fire at the Carnegie Institution of Washington and professor Craig Mello of the University of Massachusetts discovered that double-stranded RNA (dsRNA) administered to nematodes could effectively turn off genes, the era of RNA interference (RNAi) was born.

Inhibiting or “silencing” genes through chemical means is not a new idea. Antisense methods use artificial DNA that binds to and inactivates complementary target DNA sequences, thereby blocking transcription. Antisense technology has intrigued researchers since the mid-1980s because it was the first rapid, generalized technique for blocking protein expression at the molecular level.

Antisense molecule design is relatively straightforward, because scientists need only identify a critical region of the gene and create, through standard oligonucleotide chemistry, an antisense DNA strand that binds to it. Scientists know that an antisense oligo with the sequence TAACCG will always bind to complementary “sense” strands with the sequence ATTGGC.

What is RNAi?
RNAi and antisense are similar in that both use oligonucleotide sequences complementary to a molecular gene target. However, instead of binding to DNA in the nucleus, RNAi inactivates mRNA, the immediate precursor to protein, in the cytosol. When mRNA is treated with the appropriate RNAi agent, protein synthesis stops, producing an effect identical to that if the gene itself had been silenced.

Originally, RNAi was thought to arise only from long, double-stranded RNA (dsRNA). As recently as the late 1990s, RNAi was merely an interesting biological mechanism limited to such lowly creatures as C. elegans and zebrafish. In addition, the effect was short-lived and suffered interference from a competing dsRNA pathway known as the interferon response.

RNAi’s demonstration in mammalian cells was not realized until 2001, when scientists learned that the interferon effect did not interfere with dsRNA sequences shorter than about 30 base pairs. In addition to not suffering from the interferon effect, the shorter strands, dubbed “small interfering RNA” (siRNA), showed consistently longer activity.

“RNA interference is more durable than antisense,” says Joseph Fratantoni, MD, vice president of medical affairs at MaxCyte Inc., Rockville, Md. “It gives you more time to do your experiment.”

Not your grandpa’s antisense
All gene-based techniques suffer from delivery problems. Some, like antisense, are stoichiometric. That is, one molecule of drug is required for each DNA target. Since so many cells contain antisense targets, doses tend to be large, creating toxicity problems. siRNA, by contrast, targets mRNAs which are present at very low levels in cells. Although siRNA activity technically not catalytic, a little bit of siRNA goes a long way.

Whether used as a gene-knockout method for research or as therapeutics, antisense molecules are short-acting and require repeated large doses. siRNA, by contrast, is longer-acting. In some systems siRNA-induced RNAi effects persist for as long as two weeks, which would place them among the longest-acting non-depot therapeutics if they reach that stage.

RNAi is also reversible, which holds tremendous advantages in research settings where cells can serve as their own controls before and after silencing.

The combined advantages of longer duration, reversibility, and near-catalytic activity make RNAi a much more attractive strategy than antisense for treating intractable diseases. “You could say that after just a few years siRNA has already outrun antisense, and is beginning to leave antisense behind even more rapidly,” says Klaus Lun, project manager at Amaxa Biosystems GmbH, Köln, Germany. The learning curve for siRNA work has been so short because of scientists’ experience with antisense. “When RNAi came along everyone was already at the starting line, ready to go. Mammalian RNAi techniques could not have come at a more opportune time, with so much progress occurring in genomics.”

The challenge of delivery
The biggest challenge by far for siRNA/RNAi is getting siRNA into cells. Several companies are working on this problem, many borrowing technology from antisense.

About 10 companies offer reagents for delivering siRNA. Most methods are lipid-based. One such technique, borrowed from antisense work, is liposomes—microscopic soap bubbles—that encapsulate siRNA and transport it through cells’ fatty membranes. As researchers discovered with antisense, liposomal siRNA delivery doesn’t always work as promised. “Cell-loading techniques based on liposomes create a lot of background noise,” says Fratantoni.

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Principle of siRNA-mediated RNA interference. (Source: Eurogentec)
MaxCyte’s GT cell loading system uses flow electroporation—electrical pulses—that make cell walls transiently permeable. Contrary to popular belief electroporation does not “poke holes” in cell membranes. Instead, it causes lipids in cell membranes to move apart “like opening a stage curtain,” says Fratantoni.

The other drawback with lipid-based delivery is it only works satisfactorily with standard cell lines. “If you want to do experiments in primary cells where you can analyze the biological function of genes in their original environment, it’s difficult to do with existing reagents,” says Amaxa’s Lun. Amaxa sells another electroporation-based transfection system, the Nucleofector.

“With conventional transfection methods it can take up to 48 hours before the transfected gene can be analyzed, but that’s too long for busy discovery laboratories to wait,” says Lun, who claims that Nucleofactor does the job in primary cells and cell lines in less than four hours.

Electroporation-based delivery, being nonviral, does not depend on cell division, which is a big plus with nondividing cells such as resting blood cells, neurons, and lymphocytes.

Discovery: Still number one
As a technique that knocks out gene function, siRNA was immediately exploited by pharmaceutical companies as a drug discovery tool.

“Traditional drug discovery was done by trial and error—applying materials to cells until the desired phenotype is obtained,” says Sharon Engel, director of genomic data at Compugen Ltd., Tel Aviv, Israel. “Drugs discovered in this method usually are nonspecific and have extensive side effects. With siRNA, it’s possible to identify the reason a phenotype develops, validate your identification, and attack the exact location in the cell that’s responsible for disease with minimal side effects. RNAi’s sequence specificity allows the kind of specific treatment you would demand from a next-generation drug.”

Compugen’s platform technology puts genes into clusters and then assembles them to obtain the putative mRNAs which can arise from these genes. These putative mRNAs are called transcripts of genes and the collection of all these transcripts is called the transcriptome. The firm’s collaboration with Novartis, now in its second year, involves constructing transcriptomes based on public, proprietary and third-party information. The partnership was recently extended to include the design of specific siRNA ligos for target validation.

“Having the full structure of a gene, including exon/intron construction and all alternative splice variants, enables the design of siRNA specific to the target gene and that influence all splice variants or a specific one,” says Engel. “Having the full transcriptome allows analysis of the homology of the siRNA to other genes, and prevents unintentional silencing of additional pathways. The trick is not only to know what gene sequence you wish to target, but also to know what other genes might be targeted and avoid it. That’s important because siRNA needs to be very specific. One or two mismatches may prevent it from working.”

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RNAi addresses critical points in drug development. (Souce: Sequitur)
Like other chemical gene-knockout techniques—e.g. antisense, protein nucleic acids, and Morpholinos (see sidebar)—RNAi may be used in research or as a therapeutic. The pharmaceutical industry’s first commercial application of RNAi was target validation. Through target validation, scientists block a gene suspected to cause disease and note if the disease markers disappear. Using conventional target validation methods is difficult, time-consuming, and only occasionally occurs early enough in a development program to be of any significant consequence.

Today’s siRNA technology works anywhere from 60% to 80% of the time, resulting in 90% or greater reduction in target RNA and protein levels, says Dominique Poncelet, PhD, product manager for oligonucleotides at Eurogentec SA, Liège, Belgium. “There do seem to be some resistant genes which are not silenceable, but that may be related to the design of the siRNA rather than any inherent limit to the technology. Some genes may, for whatever reason, be inaccessible due to their location or secondary structure, although theoretically folding is not supposed to be important.”

Most pharmaceutical companies use siRNA/RNAi for drug target validation to support large screening efforts, for example the Novartis-Compugen agreement. “As a validation tool, some biotechnology companies specializing in RNAi technology have already generated libraries of thousands of siRNA fragments,” says Poncelet, “and screened them in model cells for whole organisms such as C. elegans or Drosophila . Based on these platforms, some biotechnology companies—for example Sequitur Inc., Natic, Mass., and Cenix Bioscience GmbH, Dresden, Germany—are proposing complete functional genomic subscription programs to traditional pharmaceutical companies to speed up drug discovery.”

Since siRNAs exhibit all of the hallmarks for the ideal sequence-specific therapeutics, more and more biotech companies will become interested in drugs that work through RNAi, says Poncelet. He mentioned Alnylam Pharmaceuticals Inc., Cambridge, Mass., and Ribopharma AG, Kulmbach, Germany, among others, as early therapeutics players and Mirus Corp., Madison, Wis., and Ribozyme Pharmaceuticals Inc., Boulder, Colo., as delivery specialists.

”Morphing” Improves Oligonucleotide Stability

Like first-generation DNA-targeting antisense oligos, siRNA is degraded by nuclease enzymes, a fact that serious siRNA drug development must eventually address. Antisense developers reduced their compounds’ susceptibility to degradation by creating chemically modified antisense oligos, for example so-called second-generation phosphorothioates. Another approach to medicines based on small gene fragments is to alter the backbone of the molecule, as Gene Tools LLC, Philomath, Ore., has done with its Morpholino synthetic oligonucleotides. In Morpholino oligos, the sugar components of the DNA/RNA backbone has been replaced by morpholine, and the charged phosphodiester linkage has given way to an uncharged phosphodiamidate. Like antisense and siRNA, Morpholino oligos inhibit targeted mRNAs through antisense binding but do so by a mechanism that involves blocking either ribosome assembly or RNA splicing within the nucleus.

Shannon Knuth, PhD, of Gene Tools says Morpholinos are more active than their “natural” siRNA counterparts and are much less toxic than phosphorothioates. “Antisense molecules are unstable, nonspecific, and toxic,” she says. “There is no known degradation pathway for Morpholinos, and they appear to be more specific for target genes than antisense or siRNA.”

Morpholinos were developed by James Summerton, PhD, an antisense pioneer, current president of Gene Tools, and founder of AVI Biopharma, which is developing Morpholinos as therapeutics. Gene Tools’ primary customers for Morpholinos are developmental biology research laboratories.

siRNA therapeutics promise the specificity and low toxicity that antisense never delivered, and of which small-molecule are probably incapable. RNAi’s differs fundamentally from antisense and “chemical” drugs in that it is a natural, endogenous process that cells already recognize and exploit. Cells were not designed to receive small organic chemicals or to have their genes silenced by phosphorothioate antisense compounds. RNAi has been with cells through evolution.

Specificity is the key difference between siRNA and other drugs, says John Maraganore, PhD, CEO of startup siRNA therapeutic company Alnylam. “siRNA is highly selective with respect to the mRNA it degrades as long as you make sure that the target sequence is unique.”

To date most siRNA work has focused on target validation as opposed to therapeutics. But as some have pointed out, due to the very nature of RNAi if you have a tool for target validation, you may also have a drug.

Because it’s so easy to create siRNA oligos, synthesis is no longer the bottleneck in discovery as can be is with small-molecule therapeutics. For example, seven genes are are suspected as possible culprits in a disease, scientists would ideally like to test each one. Making effective inhibitors to all seven proteins encoded by those genes would take years, so researchers prefer to knock out the genes, one by one, and see which knockout is effective. Because siRNA is so easy to make, all one needs to test all seven genes is knowledge of their (or their mRNA’s) structure, and an oligonucleotide synthesizer. In fact, one could synthesize many siRNA sequences for each gene and test all of them in a fraction of the time it would take to inhibit the corresponding proteins using small molecules. The exciting part is that after the siRNA set is optimized one has not just a convenient way to knock out genes and validate targets, but a potential therapeutic as well.

The relative ease with which siRNA can be produced, relative to small “organic” molecules, should not be underestimated. What makes siRNA powerful and fast in target validation makes it even more attractive for therapeutics. siRNA combines drug and target validation in one molecule, one platform, while obviating the need for traditional medicinal chemistry. With siRNA knowledge of the gene is sufficient, but not even necessary. When the gene is known so is its mRNA sequence, and therefore the exact structure of a proposed interference sequence. Narrowing that sequence down to an optimized, 20-odd nucleotide takes work, to be sure, but not as much work as optimizing a small-molecule enzyme inhibitor.

Toxicity is the main reason siRNA is more attractive than antisense (and why, way back when, antisense appeared to hold more promise than small-molecule medicines), says Tod Wolf, PhD, president of Sequitur Inc., Natick, Mass. Toxicity in small-molecule drugs and antisense arises from lack of specificity to protein and gene targets, respectively, as well as from poorly-understood mechanism-related effects.

So far we have made siRNA drug development sound easy, which of course it is not. Woolf says that researchers must solve delivery, stability, and tissue specificity issues before anything can come of siRNA therapeutics. “Naked siRNA is not serum-stable, and free oligos tend to hone in on certain tissues. So if you want to target disease sites specifically you need a way to protect the siRNA, for example by encapsulating it with lipids. In cell culture, it’s possible to use transfection agents to make RNAi more efficient, but lipids that work in cell culture do not always work in animals.”

As Woolf says, many potential problems with siRNA have been resolved through experience with antisense molecules, for example manufacturing, toxicity, and immunogenicity.

End of history for drug discovery?
Heralding the end of history for traditional drug discovery, or proclaiming RNAi as the pharmaceutical industry’s microprocessor is undoubtedly premature. But it is awfully tempting to do so. Four years after its discovery, RNAi has changed how drug discovery scientists view genes and proteins, transformed how we view RNA and DNA, and greatly expanded methods for discovering what causes disease.

Angelo DePalma
DePalma is a freelance writer based in Newton, N.J.

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