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[SANET-MG] RNAi kills
RNAi is a genetic technique which has begun to be employed extensively
in genetic engineering. The USDA plum C5 honey sweet being petitioned
for commercial release is an example of RNAi to kill viruses. The paper
below deals with the finding that RNAi may kill. RNAi is frequently
active against organisms in widely different kingdoms such as
bacteria-man, etc suggesting that RNAi genetic applications require full
safety testing of the RNAi.
Nature Reviews Drug Discovery 5, 528-529 (July 2006) | doi:10.1038/nrd2104
Safety concerns raised over RNA interference
Top of pageAbstractHigh doses kill mice, but researchers maintain
confidence in gene-silencing method
Warnings: As more is understood about the gene-silencing capabilities of
RNA interference, more issues are being raised about potential safety
concerns in therapeutic applications.
Gauging the progress of the next big thing in science is often
difficult, as failures are often greeted with similar, if not greater,
fervour as successes. No one doubts that the current flavour of the
month, RNA interference (RNAi), is likely to be a scientific revolution,
but in recent months several alarms have been sounded over how
successfully it can be developed into safe and efficacious treatments.
The latest warning call is a paper showing that mice died from liver
toxicity after being injected with high doses of a certain type of RNA
(Nature published online 25 May 2006 doi:).
This latest study has generated a lot of heat, although delegates
attending an international gene therapy meeting the week after the paper
was published remarked that the study generated a stronger response in
the media than the RNAi community. "People in the field understood that
this wasn't any kind of show-stopper — if anything, it offered further
information to move things forward," says Mark Kay, Professor of
Pediatrics and Genetics at Stanford University, and lead researcher in
the latest study.
The study should, however, raise awareness of toxicity issues associated
with RNAi. "The surprise wasn't that you got toxicity — the surprise was
what the toxicity was," says Kay.
The emergence of RNAi as a possible novel form of therapeutic has been
nothing short of meteoric. It has only been 5 years since scientists
found that silencing the activity of genes with short sequences of RNA
complementary to a gene's sequence could be carried out in mammals
without triggering the endogenous immune response to double-stranded
RNA. In that time, RNAi treatments based on introducing small,
synthesized pieces of RNA called small interfering RNA (siRNA) have
already entered clinical trials (see table).
But like most areas of drug development, the more we discover about
RNAi, the more we are beginning to understand its limitations. Studies
have revealed that some siRNAs can silence genes other than the intended
target and can activate the interferon defence mechanism (see Nature
Rev. Drug Discov. 2, 763–764; 2003). Now, Kay's group has shown that
another form of RNAi-based therapeutic — one that is more like gene
therapy, in that it packages genes encoding RNA molecules called short
hairpin RNAs (shRNAs) into viruses — also raises potential safety concerns.
Kay's team found that within a week or two of injecting their
experimental RNAi therapy for hepatitis B virus into mice, most animals
began to die of liver toxicity. According to the paper, the source of
the toxicity doesn't seem to be the RNA sequence or the virus, but
rather the dose. The original promoter sequence used for driving
production of shRNA created large amounts of RNA that overwhelmed the
RNAi system. Switching to a promoter that made fewer RNA molecules
reduced toxicity dramatically.
John Rossi, Chairman and Professor of the Division of Molecular Biology
at the City of Hope's Beckman Research Institute in Duarte, California,
tells a similar story about his studies on an shRNA-based therapeutic
for HIV. Rossi found that high levels of shRNAs are toxic in tissue
culture cells, but that a different promoter system can circumvent this
problem. "Like previous safety issues with RNAi, all of this points to
the need to optimize your dose and the fact that careful, controlled
studies are needed to assess toxicity," says Rossi. "You've got to be
careful when you are using any component of RNAi. It's an endogenous
system that can be saturated at a variety of different levels." Rossi's
team manipulate haematopoietic stem cells from a patient ex vivo, and
this, says Rossi, would detect any toxicity issues with the treatment
before the cells are re-introduced into the patient.
Irvin Chen, Professor of Microbiology, Immunology & Molecular Genetics
at the University of California, Los Angeles, has also found toxicity
with an shRNA directed against CCR5 for HIV therapy. But reducing
toxicity with a less efficient promoter also reduces potency, he says.
Finding better treatments therefore involves screening numerous RNA
sequences to identify those that strike the best balance between potency
and toxicity. "There is no way of predicting whether an shRNA is going
to be toxic or not, or potent or not for that matter," says Chen.
Dose-dependent: Mice given RNA interference therapy died of liver
failure resulting from the saturation of the RNAi process.
Chen has also found that toxicity occurs in primary cells, but not in
established cell lines. "It emphasizes the fact that it is important to
look in a system that is as close to the real life situation as
possible," says Chen. Rossi agrees. "Most people are doing cell-culture
work, and cells in culture are pretty hardy compared with primary
cells," he says.
Why and how RNAi causes toxicity isn't fully understood. One proposal is
that the lethal shRNAs compete with endogenous RNA sequences called
microRNAs (miRNAs) for the same cellular machinery that allows the
RNAi-mediated silencing process to take place. The prime suspect,
according to the authors of the latest study, is the protein exportin 5,
which shuttles RNAs into the cytoplasm, and is thought to be a
rate-limiting step for shRNA and miRNA processing. High levels of shRNAs
saturate exportin 5, preventing miRNAs that are vital for normal
cellular development from binding. Kay stresses that he isn't certain
that exportin 5 is the cause of the toxicity, and his team is now trying
to overexpress and silence exportin 5 in the liver to see how it affects
The big question is whether the siRNA-based treatments that are in the
clinic could have the same risk. In theory, this shouldn't be the case
because siRNAs enter the RNA-processing machinery downstream of exportin
5. Kay says that it is still worth looking to see whether siRNAs are
toxic in this manner, though he concedes that it is difficult to get
equivalent amounts of siRNAs into cells in a mouse liver to answer that
According to John Maraganore, CEO of Alnylam, researchers at the company
have never seen this effect with siRNAs. Maraganore says that a study in
more than 50 mice showed that siRNAs targeting the apolipoprotein B gene
in the liver and jejunum produced no effect on endogenous miRNA levels,
despite silencing the target mRNA by around 80% (Nature 432, 173–178;
2004). When the same siRNA was tested in primates, transient and
reversible liver injury were observed (Nature 441, 111–114; 2006), but
Maraganore says this seems to be related to the silencing of
apolipoprotein B, rather than the type of toxicity observed with the
shRNA study. "In our experiments we have never seen an effect on miRNAs
as it relates to the use of siRNAs," says Maraganore.
Kay is quick to point out that he still has confidence in RNAi, and
hopes that his findings do not stall the development of RNAi-based drugs
because researchers divert their energies towards investigating the
mechanisms underlying RNAi toxicity. "Like any drug, if we had to
understand total mechanism, we wouldn't have any drugs on the market,"
says Kay. "So mechanistic studies must go on in parallel, and any new
idea gives you a rationale for redesigning your strategies and approaches."
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