For Stanford Scientists, RNAi Gene Therapy Takes Two Steps Forward, One Step Back.

STANFORD, Calif. -- Three years ago Mark Kay, MD, PhD, published the first results showing that a hot new biological phenomenon called RNA interference was an effective gene-therapy technique in mice. That finding kicked off an RNAi gene therapy research flurry amongst both academic and industry research groups.

Now, with three human RNAi gene therapy trials under way, Kay's initial excitement is proving to be on target. However, reaching this point hasn't been without challenges. In the latest twist, Kay, professor of genetics and of pediatrics at the Stanford University School of Medicine, and postdoctoral fellow Dirk Grimm, PhD, report an unexpected side effect of another type of RNAi gene therapy not on trial -- mice in that study suffered liver toxicity from the treatment and some died. Despite that initial result, to be published in the May 25 issue of Nature, Kay and Grimm went on to find a way that shows promise in resolving this side effect.

"Just like any other new drug, it is just going to mean that we need to proceed cautiously," Kay said.

In traditional gene therapy the inserted DNA produces a gene to replace one that carries a mutation. In hemophilia, for example, the inserted gene makes a protein that is missing in the blood of people with the disease. RNAi gene therapy has the opposite effect. The inserted DNA produces a molecule called an shRNA, which turns off an overactive gene.

With key genes shut off, viruses such as hepatitis B, hepatitis C or HIV are unable to multiply and cause disease. However, some reports had suggested that RNAi gene therapy might induce an immune reaction or switch off the wrong gene or genes.

As these concerns faded, things began looking up for RNAi with three RNAi therapies now in human trials -- two for macular degeneration and one for a type of pneumonia. However, these studies involve simply infusing the RNAi molecules into the eye or lung. The RNAi effects in these therapies aren't permanent. Instead, patients may need to receive repeat doses of the RNAi.

If RNAi is going to be viable as a therapy for organ-wide diseases such as hepatitis B or C, it will have to stick around. Kay and Grimm felt they needed to show that the shRNA molecule made by the therapeutic gene would continue to be safe if it existed in high levels in a tissue over long periods of time.

Instead of proving the safety of RNAi gene therapy, the pair found that persistent, high levels of the shRNA made the mice sick, and in some cases the mice even died.

The problem, it seems, is that in the process of shutting down the viral genes, therapeutic shRNA molecules also hijack the cell's normal RNAi machinery. With that machinery otherwise engaged, it's not available to carry out its normal role in the cell.

"One benefit of RNAi gene therapy is that it uses the body's own machinery, making it an effective approach," Kay said. "However, the detriment of RNAi gene therapy turns out to be that it uses the body's own machinery."

Nonetheless, Grimm and Kay bypassed the toxic effects by producing the therapeutic shRNA molecule at lower levels. They were able to prevent the human hepatitis B virus from replicating in mouse liver for more than half a year after a single therapy using this technique. Kay and Grimm said they have more work to do to learn the best way of making shRNA at levels high enough to be effective as gene therapy but low enough to avoid toxicity in humans.

Kay said that cancer and viral diseases such as AIDS or hepatitis B and C are likely targets for future RNAi therapies. In order to get to these trials, Kay said he and Grimm would need to work out what caused the toxic effects in mice and further develop strategies for circumventing that reaction. He expects that trials already under way will help him and others figure out the best way to bring RNAi gene therapy safely to humans.

Postdoctoral fellows Konrad L. Streetz, PhD, and Catherine L. Jopling, PhD; research assistants Theresa A. Storm and Kusum Pandey, and veterinary pathologist Corrine R. Davis, DVM, PhD, contributed to the study.

For more information on RNAi, please visit http://mednews.stanford.edu/stanmed/2005winter/rna.html.

Stanford University Medical Center integrates research, medical education and patient care at its three institutions -- Stanford University School of Medicine, Stanford Hospital & Clinics and Lucile Packard Children's Hospital at Stanford. For more information, please visit the Web site of the medical center's Office of Communication & Public Affairs at http://mednews.stanford.edu.