Knockout punch: the promise of RNAi

Deep in your DNA, a gene has gone haywire and is driving up the production of a protein that is messing with your body. Wouldn’t it be great to sift through all your 20,000-something genes, find the offender, and swat it like a fly?

Fortunately, a new technique eventually could do just that, targeting that gene and only that gene, knocking it out of operation and relieving your distress with zero side effects.

That’s the audacious theory, anyway, behind the medical application of RNA interference (RNAi).

When RNAi emerged on the research scene five years ago from experiments at Whitehead and other labs, it was hailed as a critical breakthrough for science. The approach involves delivering tiny strands of RNA into target cells. These strands interfere with the messenger RNA molecules that control protein production and hence gene expression, giving scientists the power to knock out individual genes at will.

Now a vital tool for genomic exploration, RNAi also promises to create new drugs that would target the genetic roots of disease.

Several classes of RNAi-based drugs are at advanced stages of development. One—a treatment for age-related macular degeneration (AMD) of the eye—is in Phase 1 clinical trials. Other RNAi-based drugs still in pre-clinical development target HIV, hepatitis C, Huntington disease, and various neurodegenerative disorders.

n the race toward the clinic, RNA is at a turning point. While scientists generally remain upbeat about its clinical potential, they also are still cautious in their views. Getting the tiny strands of RNA (known as short interfering RNAs, or siRNAs) into target cells is no easy task.

Delivering RNAi compounds and ensuring that they last long enough to be useful pose continuing challenges. Some researchers doubt that RNAi will ever make it out of the lab, pointing to the decades of largely unsuccessful struggle to commercialize antisense DNA, another approach to selective gene silencing.

Preparing for trial

While working at Whitehead from 1999 to 2001, Whitehead Member David Bartel and colleagues who include Thomas Tuschl (now at Rockefeller University), Phillip Zamore (now at the University of Massachusetts Medical School), and Phillip Sharp (still at MIT) generated early insights into RNAi mechanisms in cell biology.

The group knew the research would be important for both fundamental and clinical research. “None of us had the resources or ability to develop this as a clinical tool,” Bartel says. But as pioneers in the field, with a series of influential papers behind them, the scientists and their business colleagues had little trouble securing capital for a startup. Raising $17 million, in 2002 they founded Alnylam Pharmaceuticals, which has since become a public company.

Headquartered a few blocks away from Whitehead, Alnylam focuses on diseases that are well understood genetically but have proved difficult to treat effectively with more conventional drugs. That list currently includes age-related macular degeneration (AMD, the leading cause of vision loss in the U.S.), respiratory syncytial virus (RSV), spinal cord injuries, Parkinson disease, and cystic fibrosis. Half of Alnylam’s 70-plus employees are MD or PhD researchers.

The company’s drug process kicks off by selecting a suitable disease, says John Maraganore, president and chief executive.

Second, researchers design and synthesize siRNAs that are predicted to be effective against the target. There might be 100 or 200 of these molecular candidates, with sequences chosen to work preferentially against the target gene’s messenger RNA. Another practical consideration is to choose candidate sequences that are identical between humans, mice and any other model organisms.

“Then we take the synthetic siRNAs and screen them in cell-based arrays to target reductions in messenger RNAs and proteins,” Maraganore says. “We can test a couple of hundred siRNAs within a week or so. It’s pretty common that we find 10 to 20% of them that are much more potent than the others.”

Next, the researchers screen out siRNAs that might activate the interferon reaction in cells. (RNAi molecules that look like viruses to the cell can risk being wiped out by interferon proteins that cells produce and release to the bloodstream in response to viral invasions.) Then the researchers modify siRNAs chemically to enhance their ability to pass through the cell membrane and withstand attack by enzymes within the cell. Now the survivors are ready for toxicology studies in animals, the final step before human trials.

Alnylam expects that its AMD candidate will reach human clinical trials by the end of this year, followed by its RSV drug. The pre-clinical development cycle is surprisingly fast compared to that of small-molecule or protein drug discovery, since it starts with such carefully honed targets, Maraganore says. And while human trials will follow the same procedures and schedules that they do with more conventional drugs, he hopes that success rates will be higher since RNAi follows natural pathways.

The vision thing

Here’s a similar startup story: During the late 1990s, Michael Tolentino, an ophthalmologist at the University of Pennsylvania, and colleague Sam Reich found that RNAi effectively silenced genes in the mammalian eye. The scientists proposed that RNAi could offer new treatments for AMD, a condition that occurs when the vascular endothelial growth factor (VEGF) protein becomes hyperactivated. This protein contributes to the growth of abnormal blood vessels in the eye, which leak and produce dim central vision in millions of aging patients. Other treatments have offered only limited success. Reich licensed the technology from the university, and Acuity Pharmaceuticals of Philadelphia was created in 2002.

The company’s drug development efforts have been based largely on the initial research by Reich and Tolentino. Reich—who serves as Acuity’s vice president for research and development—staffed the company with experts in basic research, manufacturing and regulatory affairs.

Acuity president Dale Pfost says that company researchers built on the university experiments with a series of in vitro and in vivo experiments designed to screen for potential drug candidates. These efforts eventually led to the isolation of Cand5, an RNAi compound that is now the company’s chief product.

The firm’s scientists then moved on to a series of pre-clinical studies designed to evaluate the drug’s absorption, distribution, metabolism and excretion in a range of mammalian species. These data, gathered in-house and by contract laboratories, must be submitted with an investigational new drug application filed with the Food and Drug Administration.

Phase 1 clinical trials with Cand5 began last fall. Pfost predicts the drug will be approved by the FDA and on the market by 2009.

Special deliveries

AMD is likely to be the first human illness for which RNAi yields approved treatments, says Irena Melnikova, a senior research analyst with Life Science Insights, a subsidiary of IDC Research. That’s because RNAi compounds can be injected directly into the eye, avoiding the systemic barriers that plague effective delivery of the compounds elsewhere in the body. Several companies are now working their own angles on the disease.

Acuity’s closest rival, Sirna Therapeutics of Boulder, Colorado, also began clinical trials late last year, with an RNA compound called Sirna-027 that targets a VEGF receptor protein rather than VEGF itself.

Compound delivery methods differ. While Cand5 is unmodified from its natural state, Sirna-027 is chemically modified to enhance its stability.

Another class of RNAi-based drugs now on the verge of clinical trials operates through a different approach often described as gene therapy––or as “expressed RNAi” because it harnesses the cell’s own genetic machinery to produce a gene-silencing response. Typically, a viral vector (viral DNA modified to carry the desired DNA) transfers instructions for making RNAi molecules directly into the cell’s genome. The cell then produces the molecules as part of a natural process.

One potential treatment based on the expressed approach targets HIV. Benitec in Mountain View, California, is creating a drug that targets a set of three genes involved in HIV. Among them, a “master regulator” called TAT controls other genes in the virus.

John Rossi, who chairs Benitec’s science advisory board, is a professor at the Beckham Research Institute at the City of Hope cancer center in Duarte, California. “We feel that with an RNAi cocktail that targets three genes, we’ll be able to keep HIV in check,” he says. Rossi, who previously worked on antisense strategies for gene silencing, is quite keen on the potential of RNAi. Benitec plans to take its compound into Phase 1 clinical trials this November.

Yellow  lights

Some researchers suggest that opportunities with RNAi may never rise beyond basic research. At best, these skeptics say, RNAi screening will speed up identification of proteins that can be better targeted with standard drugs.

Part of this skepticism comes from disappointments with previous attempts at selective gene control. For more than a decade, researchers have struggled to create successful therapies with antisense drugs that also bind to messenger RNA. Only one such drug is in use, treating certain eye infections in AIDS patients.

One issue is that antisense drugs tend to degrade rapidly, so their potency is low. RNAi proponents say that RNAi-based preparations are up to 1,000 times more active than their antisense counterparts, indicating a vastly greater likelihood of therapeutic success.

For RNAi, systemic delivery to target cells in the body remains a huge obstacle. Researchers are focusing on ways to bypass cell membranes and evade immune responses that might degrade the drugs too soon.

Alnylam made headlines with a paper, published in Nature last November, showing that RNAi compounds administered by injection could silence clinically relevant genes if they were attached to cholesterol molecules. The work demonstrated for the first time that gene silencing could be achieved in live animals through a systemic route of administration.

But dose levels were extremely high and yielded only a partial effect. The company is working to design a compound that goes directly to the target tissue and is more easily taken up.

Into the pharm leagues

For RNAi companies to succeed, they must get the blessing of major pharmaceutical companies. For the time being, says Sara Cunningham, Benitec chief executive, the major pharma companies are on the sidelines, waiting for safety and efficacy data to emerge from pre-clinical and Phase 1 research.

In the meantime, RNAi researchers are running as fast as they can.
“RNAi has enormous potential as a therapy,” says Judy Lieberman, an RNAi researcher who teaches pediatrics at Harvard Medical School.

“It’s hard to predict at this stage if it’s going to be as promising as some people might think,” sums up Lieberman. “But it’s extremely active at very low concentrations with a high degree of specificity. I think it can be used with almost any gene, so the disease opportunities are pretty unlimited. I’m very optimistic.”

Written by Charles Schmidt.

This article first appeared in the Spring 2005 issue of Paradigm magazine.