RNA Therapeutics Enter Clinical Trials

Traditional gene therapy is built on a simple premise: If the absence of a gene product causes disease, then adding the missing gene will cure it.

Amy Adams(aadams@the-scientist.com)
Jan 16, 2005

Courtesy of John Rossi

Citing concerns over efficacy, stability, and specificity, many researchers develop localized RNAi therapeutics strategies, such as for use in the eye. A novel variantion on this approach is being developed in which a patient's blood stem cells are transfected with a lentiviral vector expressing an anti-HIV siRNA. Those cells are then reintroduced to the patient, where the hope is that the cells will propagate and develop into mature blood cells capable of fending off HIV infection.

Traditional gene therapy is built on a simple premise: If the absence of a gene product causes disease, then adding the missing gene will cure it. But recently some researchers have turned that idea upside down, using gene therapy to silence genes gone bad. The approach takes advantage of a technique called RNA interference (RNAi) to specifically destroy a targeted mRNA and thereby eliminate the resulting protein.



Much of the development in RNAi therapeutics builds on the foundation of traditional gene therapy, and with at least 15 years of research under its belt, the elder field has much to offer its descendant. Some groups are taking advantage of this experience by sticking with traditional gene therapy vectors. Beverly Davidson at the University of Iowa, for instance, uses adeno-associated virus (AAV) to deliver RNA into Purkinje cells in the brain. Expression of mutant proteins in these cells (among others) leads to neurodegenerative diseases such as Huntington and spinocerebellar ataxia.

"This virus doesn't normally go into mammalian brain," Davidson says. "When we directly injected them we had no way of knowing what kind of cells they would affect." As luck would have it, the virus infects Purkinje cells, but she admits this virus may not be ideal for all brain applications. "It's better to take the simple approach and use a virus that likes your cell," she says.

One advantage AAV offers Davidson is that it inserts its genetic payload into the host's DNA rather than transiently expressing the gene from the cytoplasm. In Davidson's experiments, the virus inserts a sequence coding for a short RNA that doubles back on itself like a hairpin (called a short hairpin RNA, or shRNA), which is then processed by the RNAi machinery to produce a functional short interfering RNA (siRNA).

Using this approach in a mouse model of spinocerebellar ataxia, Davidson found that the treatment improved muscle-coordination problems characteristic of disease.2 McCaffrey, now at the University of Iowa, calls this result significant. "AAV is probably one of the more promising vectors," he says.

McCaffrey also uses traditional gene therapy approaches in his work on hepatitis viruses, using both viral vectors and naked plasmids expressing shRNA. But unlike Davidson, who injects her vector directly into the brain, McCaffrey and Mark Kay (McCaffrey's mentor at Stanford) have struggled to get their therapy into the liver in a way that is feasible for humans. Their animal approach involves injecting large amounts of virus into the tail vein of mice, or into an artery leading to the liver. "It's efficient but probably isn't going to work for humans," McCaffrey says.


One possible solution is to eliminate the vector and inject naked RNA instead – an attractive option for pharmaceutical companies, in part because of the control this gives over what they are delivering to their patients. The downside is RNA's instability, but McCaffrey says that with modification the double-stranded siRNA can survive long enough to have limited therapeutic effect.

Scientists have hit upon several effective chemical tweaks, including phosphorylation to protect against exogenous degradation, and methylation or fluorylation of the vulnerable 2' carbon to prevent endonuclease activity. "This allows us to stabilize the siRNA without loss of function," says John Maraganore, president and CEO of Cambridge, Mass.-based Alnylam Pharmaceuticals, which develops RNAi therapeutics.

The first RNAi-based therapy to go to clinical trial will actually do so without any modifications at all. Researchers at Acuity have found in mice that naked RNA survives long enough in macular-degeneration models to effectively shut down the excess VEGF protein that drives the disease. The company is now recruiting patients for a Phase I human clinical trial. Sam Reich, Acuity's senior director of research and development, says the entire testing regimen should take about four years, meaning the company could have a marketable drug sometime in 2009.


The decision by both Acuity and Sirna to target macular degeneration was certainly not accidental. The eye is a closed system, and Maraganore says naked RNAs will probably work best for localized illnesses in which the siRNA can be delivered directly to the target cells. For macular degeneration, that means injecting shRNA directly into the eye; in Parkinson disease, the nucleic acid is injected into the brain. Injections to both of these regions are already standard medical practice.

Other therapeutic strategies also may work best locally. Though proponents tout its surgical precision, RNAi's specificity is open to debate. Using microarrays to assess nonspecific inactivation, for instance, one paper found inhibition of only the targeted gene,3 while two other groups observed non-specific effects.4

And then there's the question of whether systemic RNAi induces an interferon response. Though some researchers have made observations to the contrary, Mark Davis and colleagues at the California Institute of Technology recently failed to detect an interferon response when administering siRNAs through either the tail vein or intraperitoneally.5

Beyond these questions, many of the issues surrounding RNAi therapies, including questions of stability, dosage, and targeting, can be avoided if researchers employ local administration strategies instead of systemic ones.

Adopting the local approach, Davidson delivers her AAV-based therapy directly to the brain. Hitting the protein in all tissues may cause unexpected side effects, she says, especially if the treatment eliminates both the normal and mutated protein. "I would be concerned about off-target effects if it was being used systemically," she says.


Not everyone is committed to going local. Last November Alnylam described a way to deliver RNAi therapy both systemically and effectively, attaching its siRNA to cholesterol to ferry the molecule through the bloodstream.6

"It was an experiment that seemed unlikely," says senior author Hans-Peter Vornlocher. "It was unbelievably exciting." Vornlocher is also vice president of research at Alnylam Europe (previously Ribopharma), based in Kulmbach, Germany. The siRNA targeted apolipoprotein B, a key regulator of cholesterol metabolism whose levels correlate with an increased risk of cardiovascular disease. Mice receiving the modified siRNA had a statistically significant reduction in ApoB protein (37%), high-density lipoproteins (25%), and low-density lipoproteins (40%).

In an accompanying review article, John Rossi at the City of Hope Beckman Research Institute in Duarte, Calif., called the study a big step for RNAi therapy. In a separate interview, Rossi says, "Clearly this is the first published demonstration that you can systemically inject siRNA and have them be taken up by cells in several different tissues."

The cholesterol conjugate used in the study carried the siRNA to organs including the liver and jejunum, where most ApoB is produced. But other conjugates may target the therapy elsewhere, to organs such as the liver or kidney, says Rossi. "This is just the beginning. Other investigators are very interested in looking at a wide range of alternate conjugates," says Maraganore.

Rossi's own work takes another novel approach to RNAi therapy. Rather than trusting the siRNA to makes its way to the correct tissue – either as naked RNA or in a viral vector, Rossi transfects blood stem cells with a gene coding for an anti-HIV shRNA.7 He plans to reinject those stem cells into patients, where they will mature into immune cells that produce HIV-thwarting siRNAs. Rossi hopes to use this approach in combination with other therapies in patients with HIV and lymphoma who need a bone marrow transplant after radiation therapy. "By treating these and giving them lifetime protection from the virus, you might be able to take people off some of the chemotherapy," Rossi says.

None of the techniques currently being investigated yet rises to the level of a magic gun. "Excitement has to be tempered with reality," says Rossi. His therapy, for instance, would require gram quantities of conjugated siRNA to work in humans. Nevertheless, all the strategies have shown promise in early studies. With a little luck, any of these could develop into a delivery device worthy of carrying the RNAi magic bullet.