ISIS Report 12/07/06
Gene Therapy Nightmare for Mice
Could Humans Be Next?
therapy ‘breakthrough’ in precision turns out to have many off-target effects
and killed more than 150 mice. Time for
gene therapist to take a system’s view of genetics and biology before proceeding. Dr. Mae-Wan Ho
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A gene therapy ‘breakthrough’
in precision touted by medical researchers for treating HIV, cancer, neuro-degenerative
diseases, hepatitis, and more, was found to have many off-target effects a
year ago in 2005  (Controversy over gene therapy ‘breakthrough’,
SiS 26), raising considerable concerns over
The technique is
based on RNA interference (RNAi), using short stretches of RNA to target genes
in a sequence-specific manner, in theory, and silence them. Unfortunately,
many other genes and proteins were affected, the precision was illusory. Undaunted,
proponents carried on, hoping that the off-target effects would be addressed
by further research.
Then in May 2006, the nightmare unfolded. RNAi gene therapy
ended up killing mice by the dozens [2, 3]. The finding came from the laboratory
of gene therapist Mark Kay of Stanford University California,
USA, whose research team reported 3 years ago that RNAi inhibited the hepatitis
B virus in mouse livers.
The team has administered
a refined version of the RNAi treatment to infected mice, using short hairpin
RNA (shRNA), a precursor to the microRNA (miRNA) species  (Subverting the genetic text,
SiS 24) previously used. For the first couple
of days, everything was as expected. But within a week or two, the mice began
to fall ill, their skin turning yellow from liver damage. More than 150 animals
died, and many others suffered liver toxicity. Kay and postdoctoral fellow
Dirk Grimm, while taken aback by the toxicity of the treatment, said they
and others still had confidence in RNAi. “I really think it can still work,”
It has better work,
because companies have been testing RNAi on people for treating a respiratory
virus and macular degeneration since October 2004.
that we don’t understand going on here”, said Timothy Nilsen, head of the
Center for RNA Molecular Biology at Case Western Reserve University Cleveland,
Kay’s team packaged
genes encoding the shRNA molecules into viruses stripped of other genetic
material, and injected the viruses into the mice. The viruses then infected
the cells and kept producing the shRNAs, thus making a single dose go much
further. The virus used was an adeno-associated virus (AAV) that homes in
on the liver, and sure enough 90 percent of the virus-delivered genes ended
Is the virus to blame?
Apparently not, there were no problems injecting ‘empty’ virus without the
Was it the shRNA?
Kay’s team created dozens of viruses making other RNA sequences and injected
those into mice without hepatitis B. Out of
all 49 sequences, 36 were severely toxic; 23 were lethal in every case, killing
the animals within two months. So the effect had nothing to do with
any specific gene targeted by the shRNA.
Was it an overdose
of small RNA? Kay’s team found that death was associated with low levels of
the mice liver’s own miRNA, which are necessary to the liver’s function, indicating
that perhaps the shRNA injected was competing for processing or transport
of the small RNAs. The mice were dying from liver failure possibly from an
overload of shRNA in their livers. The team had apparently safely inhibited
the hepatitis B in mice by injecting an AAV that made less RNA.
John Rossi of
City of Hope in Duarte California, who is working on RNAi therapy
for HIV, said the results were “not surprising in retrospect”. Too many extra
RNA molecules may disrupt the cells’ own RNAi machinery. Kay’s group suggested
that the extra small RNA compete for a protein, exportin-5, that transports
the cell’s own small RNAs out of the nucleus.
setbacks and warnings, a company called Sirna Therapeutics in San Franscisco
California is still planning to test a nonviral RNAi strategy
on people with hepatitis C next year. Sirna’s chief scientific officer Barry
Polisky said that the company “has spent a hell of a lot of time and effort”
putting small RNAs into animals and nonhuman primates looking for toxicity
and haven’t seen anything like what Kay’s team has found.
But Nilsen for
one is not convinced. “I think it is premature to say anything is safer at
this point”, he said.
People in the field understood that this
wasn’t any kind of show-stopper – if anything it offered further information
to move things forward,” Kay said .
The only reason to cling
to the belief that current RNAi therapy in humans is safe is that Kay’s group
has used shRNA rather than miRNA, which is downstream of shRNA. But if shRNA
competes for limiting amounts of a protein exporting the small RNA out of
the nucleus, then it would happen whether miRNA or shRNA is used.
In my opinion, RNAi
gene therapy is unsafe on the whole because the effects are not, and cannot
be specific, even more so than conventional DNA gene therapy  (Gene therapy woes, SiS 26). Numerous RNA species interfere at
every level of gene function  (Subverting
the genetic text, SiS
24), and it is impossible to target the effects
precisely because the RNA interference underworld is huge, comprising some
97 to 98 percent of the transcription activity in the cell, and the specificity
depends on low levels of the correct sequences being produced at the right
time in the appropriate places in a dynamic molecular consensus, ‘a dance
of life’ that’s the essence of the fluid genome  (Living with the Fluid Genome).
Gene therapist should
really take an appropriately holistic and systems view of genetics and biology
before they create more diseases than they cure.