RNAi Safety Comes Under Scrutiny

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MOLECULAR BIOLOGY:
RNAi Safety Comes Under ScrutinyJennifer Couzin
What began as an effort to craft a better hepatitis therapy using a strategy called RNA interference has ended in the deaths of dozens upon dozens of mice--a harsh safety alarm for biomedical researchers looking to RNAi as a treatment for HIV, cancer, neurodegenerative diseases, and more.

The results, from gene therapist Mark Kay of Stanford University in California, come 3 years after he reported that a treatment based on the gene-silencing technique inhibited replication of the hepatitis B virus in mouse livers. This time around, Kay's team administered a refined version of the RNAi treatment to more than 50 infected mice.

\"We saw for the first couple days exactly what we expected,\" says Kay's postdoctoral fellow Dirk Grimm, who helped lead the studies. But within a week or two, the mice began falling sick, their skin turning yellow from liver damage. More than 150 animals died, and many others suffered liver toxicity. Lowering the amount of virus given eliminated the harsh effects but also erased the treatment's success.

\"There's something that we don't understand going on here,\" says Timothy Nilsen, who heads the Center for RNA Molecular Biology at Case Western Reserve University in Cleveland, Ohio. Although Kay and Grimm were taken aback by the devastating toxicity, they and others retain confidence in RNAi. \"I really think it can still work,\" says Kay.

RNAi has become enormously popular in the last few years. It involves blocking the activity of genes, including those linked to disease, with short sequences of RNA complementary to a gene's sequence. Companies are already testing in people RNAi treatments for a respiratory virus and for macular degeneration.


Interference problem. Compared to the liver of a healthy mouse (above), an RNAi treatment destroys the liver of a treated animal (below).
CREDIT: M. KAY/STANFORD UNIVERSITY


Those trials, for which no significant safety problems have been disclosed so far, rely on simply introducing RNA molecules into the body. In contrast, Kay's team packages genes encoding small RNA molecules into viruses stripped of other genetic material, a strategy much like traditional gene therapy. Once injected, the viruses infect cells and keep producing the small RNAs, allowing a single dose to go a long way.
For its RNAi tests, Kay's team uses an adeno-associated virus (AAV), which homes to the liver. Indeed, 90% of the virally delivered RNA genes ended up there, says Grimm. Yet the virus is probably blameless; injections of an empty virus didn't cause problems in the mice. To explore whether specific RNA sequences might be the culprits, the Stanford team created dozens of viruses making other RNA sequences and injected them into mice without hepatitis B, some genetically altered and some normal. Out of all 49 sequences tested, 23 were lethal in every case, killing the animals within 2 months. Another 13 were \"severely toxic\" to the liver, they write in Nature. As with many treatments, dosing seems to correlate with risk: Kay's team safely thwarted hepatitis B in mice by injecting an AAV that makes fewer RNA sequences.

The results are \"not surprising in retrospect,\" says John Rossi of City of Hope in Duarte, California, who's working on an RNAi therapy for HIV. Too many extra RNA molecules may disrupt a cell's own internal RNAi machinery, he explains. Kay's group suggests that the extra small RNAs compete for a protein that transports a cell's own RNAs.

A company called Sirna Therapeutics in San Francisco, California, still plans to test a nonviral RNAi strategy on people with hepatitis C next year. The firm \"has spent a hell of a lot of time and effort putting [small RNAs] into animals and nonhuman primates … looking for toxicity, and we haven't seen anything like this,\" says Barry Polisky, Sirna's chief scientific officer. Like Kay and others, Polisky worries that these new findings will be seen as an indictment of RNAi therapy, even though he is confident that injecting small RNAs alone is less hazardous than the viral approach. Not everyone's convinced. \"I think it's premature to say anything is safer at this point,\" says Nilsen.