Mice Deaths Are Setback in Gene Test
By Andrew Pollack
New York Times
May 25, 2006
A large number of mice died unexpectedly in a test of a new technique for inactivating genes that has been widely proclaimed a breakthrough, scientists are reporting today.
The finding could give rise to new caution about the technique, called RNA interference, which is already widely used in laboratory experiments and is starting to be tested in people as a means of treating diseases by silencing the genes that cause them.
But Dr. Mark A. Kay and colleagues at the Stanford University School of Medicine report today in the journal Nature that the technique, also called RNAi for short, caused liver poisoning and death in mice.
"It's a very striking result — all of the fatalities observed and the toxicity, which was unexpected," said Timothy W. Nilsen, director of the center for RNA molecular biology at Case Western Reserve University in Cleveland. "It's really a note of concern for rapid therapeutic development of RNAi."
But Phillip Zamore, an RNAi expert at the University of Massachusetts, said the Stanford scientists had used a variation of the technique that was "no longer state of the art" and required a very high dose. The tests already conducted in people involve a different technique and lower doses, said Dr. Zamore, who is a co-founder of Alnylam Pharmaceuticals, a company in Cambridge, Mass., that is developing drugs using RNAi.
Dr. Kay himself said he thought the findings were "not a showstopper by any means" for the field. "It's like any drug," he said. "The toxicity depends on the dose."
RNA is the chemical cousin of , which encodes hereditary instructions in genes. RNA was once thought to be a mere messenger in the cell. But in a rush of discoveries over the last few years, scientists have found that RNA plays a more active role in controlling gene activity.
They have found that cells make tiny snippets of RNA, called microRNA, that silence particular genes. And they have learned how to harness that natural mechanism to turn off any gene of their choosing by inserting the proper piece of RNA into cells.
But Dr. Kay said his experiment showed that if too much interfering RNA was put into a cell, it could overtax the cell's ability to process its own microRNA. "The good news about RNAi is that it uses the cell's machinery to do its work, and that is why it's so effective," Dr. Kay said. "The bad news about RNAi is that it uses the cell's machinery to do its work. If you overload the system, you hijack the machinery from performing its normal duties."
Dr. Kay and his team, led by a postdoctoral researcher, Dirk Grimm, wanted to cure mice of B, not to kill them. They induced RNA interference in the mice's liver cells, intending to silence one of the hepatitis major genes.
In some cases this worked, and the virus was suppressed without side effects. In other cases the mice got liver poisoning, and some of them died.
The researchers tried the same thing in mice without hepatitis and then they tried using RNA interference to turn off different mouse genes.
Of 49 different RNA snippets meant to shut down six different genes, 36 caused liver injury and 23 led to death within two months. The RNA at the highest concentration was most toxic.
But some scientists said the problem appeared to be related to the technique used by Dr. Kay, which is a type of gene therapy. The Stanford team put DNA, not RNA, into the mouse liver cells. The DNA became part of the mice's genetic makeup and the mouse cells then produced the gene-silencing RNA, which was carried out of the nuclei into the body of the cells.
The scientists hypothesized that a protein that transports RNA out of the nucleus became overloaded.
The other technique is to put the interfering RNA itself into cells. That RNA does not need to be transported out of the cell nuclei, so that transporter protein would not be overloaded, some scientists said.
"These data really represent the fundamental limitations of gene therapy, not of RNAi," John Maraganore, chief executive of Alnylam Pharmaceuticals, said of the report in Nature. The company has already reported that it safely tested a nasal-spray RNA drug intended to treat a respiratory infection, on 65 healthy volunteers.