John Earle/Alnylam DRUG DESIGN Alnylam scientists are engineering better lipid nanoparticles.

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Just a few years ago, it was easy to capture the attention of a pharmaceutical industry enthralled by the therapeutic potential of RNA interference (RNAi). All an academic lab or small biotech firm needed to do was to figure out how to deliver small interfering RNA (siRNA), the key double-stranded molecule in this gene-silencing pathway, to cells. Even with just a little data showing the delivery technology worked in a test tube or tissue culture, funding was almost guaranteed.

The definition of success has changed since then. Pharmaceutical and biotech companies have invested hundreds of millions of dollars to develop or acquire RNA interference-based therapeutics, and they are demanding true validation of delivery technology. They want to see evidence that a new technique works in mice or, better yet, monkeys, and that it can be reproduced. After all, the promise of RNAi-based therapeutics hinges on it.

“Five years ago, if you had knockdown of a gene by delivering your agent to a rodent, you could write a nice paper and everyone would be happy,” says Barry Polisky, chief scientific officer of Bothell, Wash.-based MDRNA, one of several biotech firms betting on RNAi-based drugs. “Now, that’s no longer where the bar is set. And that’s a good thing.”

Scientists are working hard to transition their research from the benchtop to mice, primates, and humans. But getting siRNA to work as a therapeutic is trickier than many in the field had anticipated; simplistic notions of drug delivery have given way to focused efforts to solve the delivery problem.

RNAi-based therapeutics tap into a natural pathway cells use to control gene expression and defend against attack. Under normal conditions, single-stranded messenger RNA carries protein-building instructions from DNA in the cell’s nucleus out to the ribosomes for translation. Viruses try to sneak their own long, double-stranded RNA instructions into this process. In defense, cells use an enzyme called Dicer to chop any double-stranded RNA into pieces 20 to 25 nucleotides in length. Those small bits then bind a protein complex known as RISC, which keeps the viral RNA from doing damage.

The double-stranded siRNA molecule uses the same pathway to control gene activity. To turn off production of a protein, siRNA, also generally 20 to 25 nucleotides long, binds the messenger RNA of the protein to be turned off and guides it to RISC to prevent translation of its message.

In the early days, scientists naively thought they could just send naked siRNA strands into patients and knock down a target gene. Except for topical or direct delivery to places like the lung, eye, and the central nervous system, that facile approach hasn’t panned out. Researchers have had to come up with more involved delivery approaches instead.

“Just as all roads lead to Rome, all roads lead to cellular delivery,” says Steven F. Dowdy, Howard Hughes Medical Institute investigator and professor of cellular and molecular medicine at the University of California, San Diego, Medical Center. “Today, everybody in the field recognizes that delivery is the problem to solve, and all other problems, of which there will be numerous ones, pale in comparison.”

Scientists trying to deliver siRNA need to engineer around several troublesome properties. RNA has a molecular weight that is 10 to 20 times that of a traditional small-molecule drug. And because the molecule is highly negatively charged, it typically can’t cross the similarly negatively charged plasma membranes to enter the cell. It’s no wonder naked strands of siRNA didn’t make it as a therapeutic approach.

Delivery systems for siRNA must overcome three major obstacles: getting the drug to its target in the body, coaxing it inside the cell, and releasing it. Even after all that is accomplished, companies then need to worry about safety, a major concern given the power of siRNA to turn off cellular processes.

Lipid- and polymer-based systems are the most established approaches for systemic delivery of RNAi. In the clinic, lipid nanoparticles (LNPs) have advanced the most. Alnylam Pharmaceuticals, widely acknowledged as the leader in the RNAi arena, has a liver cancer drug in Phase I trials that applies Tekmira Pharmaceuticals’ stable nucleic acid lipid particle technology. Alnylam is also conducting early-stage studies of other drugs that use its own LNP formulations.

Some people consider lipid-based particles as old, creaky delivery vehicles. Indeed, liposomes, or tiny spheres formed by lipids, have been used for decades to encapsulate and solubilize hydrophobic small molecules or other drugs.

But while all liposomes have common features, namely materials that mask the charge and size of the molecule they deliver, not all systems are created equal, points out Akin Akinc, associate director of research at Alnylam. “We’re asking them to do something very different,” he says. A particle containing siRNA first needs to be internalized by the cell; once it gets into a vesicle called the endosome, it needs to avoid being shuttled to the lysosome, where it would otherwise be broken down; finally, it has to deliver material to the cytoplasm of the cell. “What we’re trying to do is a big challenge; we’re trying to do what the cell has evolved to prevent from happening,” Akinc says.

Alnylam’s LNPs contain multiple components: cationic or fusogenic lipids, which interact with bilayer membranes; polyethylene glycol, which creates a steric shell around the particle to lengthen its time in circulation; and lipids naturally found in biological membranes, like cholesterol or phosphatidylcholine, which provide structure to the particle.

“Just as all roads lead to Rome, all roads lead to cellular delivery.”

When constructing particles, “everything is important,” says Mark J. Murray, chief executive officer of Tekmira. Among the considerations are lipid composition, the relative amounts of the various lipid components, particle size, getting a particle to circulate faster or slower through the bloodstream, and reaching other tissues. “It’s multifactorial,” Murray notes.

“Very small changes in the identity of those components, the chemistry, can have a dramatic impact on activity,” Akinc adds.

For both LNPs and polymer-based systems, the cationic component is critical: It enables encapsulation of siRNA and provides the particle with a net positive charge that allows it to interact with cell-surface molecules. Researchers spend a lot of time designing the cationic lipids or polymers because too much of a positive charge will cause the particles to accumulate in the blood, creating toxicity; not enough charge, and they can’t be internalized by the cell.

One bright spot is that once researchers figure out how to get an LNP or polymer system into a particular tissue, the possibility opens for treating a host of diseases; in general, companies need only to change the siRNA payload.

So far, most of the success with both LNPs and polymer-based systems has been in delivering siRNA to the liver, which has leaky walls that enable the particles to slip in, explains Jon Wolff, vice president and head of research at Roche’s Madison, Wis., labs. Tumor vasculature and blood vessels are similarly “leaky,” and both are the subject of intensive drug development efforts, he adds.

Currently, the drug industry is focused on designing newer and better lipids that could enhance delivery to different kinds of tissues. As a result, scientists have become better at getting the particle inside the cell, but getting the siRNA out of the pathway to the destructive lysosome remains a major hurdle. “If the particle is unable to escape the endosome, it enters into a degradation pathway. Basically, it’s game over,” Akinc says.

Most delivery systems in advanced stages of development release siRNA in response to the lower pH of the endosome. Researchers have synthesized lipids adorned with amine groups that are easily protonated in that acidic environment and fuse with negatively charged lipids in the endosomal membrane. The fusion disrupts the particle and allows siRNA to escape. Protonation happens quickly but not always efficiently, and researchers must still come up with better ways to release the particle’s payload.

At Massachusetts Institute of Technology, Daniel T. Anderson and Robert S. Langer Jr. are working with Alnylam to develop new methods and materials for delivering siRNA. During earlier research to study ways to deliver DNA, they found that polymers that are good at delivery are difficult to design rationally, Anderson says. In lieu of trying to divine which polymer to engineer, they instead developed a way to quickly synthesize a variety of polymers for testing. By 2003, they had created a library of polymers that could be rapidly screened as delivery agents for genetic material.

In its collaboration with Alnylam, the Langer lab has started to develop an understanding of how the structure of a lipid determines its effectiveness in a delivery vehicle. A lipid’s hydrophobic regions, the way it responds to pH changes, and its potential to hold a charge all influence its value in constructing LNPs.

“The libraries are enormous and ever-increasing,” Langer says. “It’s basically a question of time and screening to find the right target.” Indeed, the collaboration has already led to an LNP formulation that has been effectively delivered to primates and is now in preclinical development.

View Enlarged Timeline Rapid Translation Milestones in moving RNA interference out of the lab and into patients

Meanwhile, Tekmira, Alnylam’s biotech company partner, has its own internal effort to develop new cationic lipids, Murray notes. “The cationic lipid component is an important one and is an area where we have spent a lot of time and energy over the last years,” he says. To that end, the biotech firm and Alnylam have also partnered with scientists at the University of British Columbia to develop more effective cationic lipids.

Among other companies advancing LNP technology, MDRNA is focusing on amino acid chemistry. The firm is taking advantage of the three “chemistry handles” available for modification on amino acids to create a different kind of particle, explains MDRNA’s Polisky, who was the chief scientific officer of Sirna Therapeutics before it was acquired by Merck & Co.

Every amino acid contains a carboxylate group and an amino group, both of which can be easily modified with alkyl chains through amide linkages. It’s a useful alteration, Polisky says, because the linkage degrades easily in the body. The third “handle” is the head group—the organic group that characterizes each of the 20 natural amino acids. This group can be positively or negatively charged, allowing for some pH-induced control of the activity of the particle. “It’s very useful in the construction of a liposome by providing electrostatic energy,” he adds.

Now with patents under its belt, MDRNA is working through the strengths and limitations of its delivery concept, Polisky notes. As with Alnylam and Tekmira, MDRNA’s early data are based on liver diseases, but the firm is hoping to engineer its particles to access other tissues as well.

Roche is also jumping onto the LNP bandwagon. In May, the Swiss giant announced that it would use Tekmira’s technology to formulate its first two siRNA drug candidates. The goal is to file for regulatory approval to test the first candidate in humans by the end of 2010.

But Roche also has a substantial internal effort around alternative delivery systems, thanks to last year’s acquisition of Mirus Bio. Roche paid $125 million for the Madison-based biotech firm, which has one of the few delivery technologies outside of Tekmira’s or Sirna’s that has been validated in primates.

Mirus’ dynamic polyconjugate (DPC) technology uses polymers to deliver siRNA into cells. In the past, toxicity has been a stumbling block for polymer-based systems: Once the highly cationic molecules entered the bloodstream, they headed straight to the most negatively charged organ—the lung—explains Roche’s Wolff.

Mirus, now known as Roche Madison, masks the positive charge by attaching polyethylene glycol and other ligands to its polymers through linkages that are quickly cleaved once they are inside the acidic environment of the endosome. That masking “is vital,” says Jim Hagstrom, vice president of operations at Roche Madison. “We don’t want these polymers working on the untargeted cells in the bloodstream or lung capillaries. We only want them to work in a defined environment.”

The masked polymers are also linked to a targeting molecule, such as a sugar molecule that directs the particle to liver cells. Lipid formulations passively target the liver, winding up in the organ effectively without the addition of a ligand, Wolff concedes. But targeting molecules will be needed to get siRNA to diseases in many other parts of the body. To reach tumors, for example, lipid formulations will need to be significantly modified, Wolff says. “We think the basic DPC particle would be the same for most cell types, and we would just change” the targeting molecule.

Still, most of Mirus’ data are from studies with liver cells, because they continue to be the easiest target. The company has published results with mice and this month will unveil data showing its technology’s ability to silence a gene responsible for making apolipoprotein B, the main kind of “bad” cholesterol, in monkeys, Wolff says. With one shot of its drug candidate, bad cholesterol disappears from the blood for a month, he adds.

Despite promising early clinical results for LNPs and polymer-based systems, they have some well-known limitations. Drugs based on lipid delivery systems have to be injected, points out Michael Czech, a molecular medicine professor at the University of Massachusetts Medical School. Even more worrisome is their inability to be directed to tissues of interest. “Most of these things by default go to the liver, and that’s very easy to do,” Czech says. “But trying to get them focused to other tissues has proven exceedingly difficult.”

As a result, companies developing RNAi-based therapeutics are constantly scanning the landscape for fresh ways to turn siRNAs into drugs. Despite its heavy investment in LNPs, Alnylam is agnostic when it comes to delivery, and at any given time, it has partnerships with dozens of academic labs and biotech firms working on new technologies.

Merck has its own lipid-based system from its purchase of Sirna, but it is also developing other delivery systems. Hundreds of academics and small biotechs are working on the problem, and Merck has evaluated more than 350 opportunities in the delivery space, says Alan Sachs, vice president of RNA therapeutics for Merck Research Laboratories. Most are iterations of LNPs or the polymer-based approach.

The company also has found that few companies have concrete data to back up their approach. “Within those 350 opportunities, there have been maybe three that have actually made it to the point where we can replicate their claims,” Sachs says. “We’re at a point where there are a lot of really smart ideas, but many have no data.” Only about a third of the group has experimental data in animals, and just a handful has tried their approach in primates, he adds.

Merck is explicit about what it expects from potential partners; Sirna has posted guidelines for safety and potency data on its website. Though the pharma giant is more than happy to provide potential partners with chemically modified RNA to get them started, the full page of requirements sends a clear message: The bar for success is high.

Meanwhile, academic labs and small biotechs are pursuing multiple delivery programs that go beyond liposomes and polymers.

UMass’s Czech, for example, is a cofounder, along with Craig C. Mello, who won the Nobel Prize for the discovery of RNAi, of RXi Pharma, which is engineering a nanoparticle that is not an LNP.

RXi has licensed delivery technology from Czech and fellow UMass scientist Gary Ostroff that targets the macrophage, a white blood cell in the gut that swallows and digests pathogens, then tells the immune system whether to react. The idea is to feed siRNA into that process by encapsulating it in a shell—essentially a hollowed-out yeast particle—covered with β-1,3-d-glucan, a sugar that binds to the macrophage. Once the macrophage takes up the particle, its acidic environment appears to break down the shell, releasing a nanoparticle encapsulating the siRNA payload.

Czech sees several advantages to delivery by glucan shells. Macrophages have broad potential as therapeutic targets, he notes, because they play a role in several diseases, including arthritis and some autoimmune diseases. They also play an indirect role in other ailments, such as cardiovascular disease, type 2 diabetes, and metabolic diseases. Finally, they are involved in harboring bacteria and viruses, which also could be modulated by RXi’s delivery technique.

Furthermore, companies using LNPs must reconfigure the particle to reach new tissues or target new diseases, whereas the glucan particle has a single target, the macrophage. “We do not have to reengineer the glucan particle for every different indication because it will always get to the macrophage,” Czech says. In addition, RXi believes the glucan shells can be formulated for oral delivery, a more patient-friendly route than injection, particularly for chronic diseases such as diabetes or arthritis.

Czech acknowledges that RXi’s delivery technique isn’t universal because right now it can access only one cell type. But so far data from in vivo studies suggest that the method is promising and could open much broader therapeutic applications than LNPs do, he contends. The company and its academic collaborators are now trying to tweak the nanoparticles contained within the shell to promote the release of siRNA. “We’re sort of in the trenches right now working on the chemistry,” Czech says.

Others are trying to avoid the need for any kind of outer shell or nanoparticle in favor of delivering a single, soluble siRNA molecule. The idea is to access more tissues while bringing more certainty and safety to drug delivery; although nanoparticles are stuffed with siRNA, companies cannot control how many strands each particle actually delivers. “The particle can carry 100 siRNAs, and whether the cell takes up two or takes up three makes a huge difference,” UCSD’s Dowdy says.

“One would like to say the path is clear, but in reality this is still a large challenge.”

Dowdy’s work, which has become the basis for the La Jolla, Calif.-based biotech Traversa Therapeutics, seeks to deliver one siRNA at a time, while also opening up the number of cell types that can be accessed with siRNA.

His technology takes advantage of a natural process called macropinocytosis, a mechanism that all cells appear to use to swallow large amounts of fluid or other material. The idea is to bind siRNA to the peptide transduction domain (PTD), a protein fragment that controls macropinocytosis.

The domain, however, contains only eight positive charges, not enough to trump the 40 negative charges on siRNA, so the two can’t be directly linked without causing molecules to aggregate, Dowdy explains. Traversa’s solution is a fusion protein that links the PTD to another positively charged protein fragment that is noncovalently bound to the siRNA. By coating siRNA with the fusion protein, it’s charge is masked, enabling it to reach its target. Once inside the cell, the drop in pH causes the release of siRNA from its delivery vehicle.

Dowdy concedes that a lot of work still needs to be done on the system, which so far has been proven only in test tubes. However, he notes that PTD delivery is already being validated for use in delivering proteins in a handful of clinical trials. If the method works, it could break open the therapeutic possibilities of siRNA because, as Dowdy says, “The beauty of these PTDs is that they appear to go into all cell types.”

Watertown, Mass.-based Dicerna Pharmaceuticals is also working on ways to deliver single molecules of siRNA. The company was founded in late 2007 on the basis of work done by John Rossi, a molecular biologist at City of Hope’s Beckman Research Institute, in Duarte, Calif., and Mark Behlke, vice president of molecular genetics at Integrated DNA Technologies, in Coralville, Iowa. The pair discovered that not only is it possible to make functional siRNA molecules longer than 21 or so nucleotides in length, the size most RNAi work has centered on, but that these longer RNAs surprisingly also knock down a target.

It turns out that the longer strands, which Dicerna is calling DsiRNA, enter the RNAi pathway farther upstream than the 21-mers. Whether via LNPs or polymers, current approaches feed siRNA directly into the cell’s RISC complex. Dicerna’s DsiRNA, however, acts as a substrate for the enzyme Dicer, which clips it down into 21-mers before handing it off to RISC.

“Because DsiRNAs engage more fully with the gene-silencing pathway, they have taken on some properties that are quite extraordinary,” says James C. Jenson, cofounder and CEO of Dicerna. “They are much more potent than 21-mers and have a longer duration of action in the cell.”

It also means complex chemistry is not needed to prompt the delivery vehicle to release the drug. “Dicer really does that work for us,” Jenson says.

The architecture of the molecule also enables targeted treatment. “Perhaps the most important advantage to these longer DsiRNA molecules is that we have a handle on the end that Dicer clips off,” Jenson notes. Thus, Dicerna can attach a molecule such as an antibody or a peptide that zeroes in on the cell of interest. The RNA enters the cell, and the enzyme then chops off and discards the targeting molecule.

Dicerna recently teamed with fellow Boston-area biotech Archemix to link its lengthier RNA chain to aptamers, targeting molecules that tightly bind to specific cell-surface receptors. City of Hope’s Rossi recently published in vitro and in vivo work demonstrating the efficacy of the RNA-aptamer conjugate technology, and this summer he presented as-yet-unpublished in vivo data showing its efficacy at the RNA Interference Summit in San Francisco.

A flood of other delivery ideas is coming out of academic labs and small companies. Some represent small improvements over existing technology, whereas others are fairly outside the box. Big drug companies can’t ignore incremental improvements in LNPs or delivery polymers, Merck’s Sachs says, but Merck is most interested in ideas that transform the RNAi field. “It’s like the CD versus the tape,” Sachs says. A groundbreaking idea that works would “fundamentally change the playing field.”

Drugs that apply the lipid and polymer-based technologies are poised to enter late-stage trials and possibly even reach the market in the next few years. “The first success will likely be in the liver, and probably oncology after that,” Roche’s Wolff says.

More importantly, those late-stage studies will provide early evidence of the utility of siRNA as a therapy, which in turn will inform drug delivery and clinical-trial design for future generations of therapeutics. “The positive outcome of these liposomal compositions is we’ll get an understanding of what the body’s response is to RNA,” UCSD’s Dowdy says.

Beyond that, most scientists agree, more cutting-edge research and thinking will be needed to overcome the myriad obstacles to broader therapeutic use for siRNA. “One would like to say the path is clear,” MDRNA’s Polisky says, “but in reality this is still a large challenge.”