AUGUST 2006
A vitally important analysis of genetic therapy
ISIS Press Release 12/07/06
Gene Therapy Nightmare for Mice
Could Humans Be Next?
A gene 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 [1] (Controversy over gene therapy ‘breakthrough’, SiS 26), raising considerable concerns over safety.
| A gene therapy technique, hailed as
2002’s ‘breakthrough of the year’
in its ability to shut down specifically
and precisely any chosen gene, has been
found not to be so specific or precise after all.
The technique involves RNA interference
("Subverting the genetic text", SiS
24), the ability of a short specific duplex
sequence of RNA to target the transcript of gene,
thereby shutting it down. Unfortunately, there
are "off-target" effects on other genes
and proteins. The technique depends on a perfect match between the siRNA (small interfering RNA) introduced and its complementary sequence in the gene transcript. Only sequences of 19-21 base pairs are generally used, as longer sequences induce nonspecific immune reactions. However, various mismatches between the siRNA and its target appear to be tolerated, so that other transcripts with similar sequences are also affected. Peter Linsley, executive director of cancer biology for Rosetta Inpharmatics, a company based in Seattle, Washington, USA, and a subsidiary of drug giant Merck, was using siRNAs to design more targeted drugs. The plan was to use siRNA to shut down a particular gene, and then add a compound that also targets the gene to see if additional genes are affected. But his team found that the siRNAs were shutting down more genes than just the one intended. "The siRNAs were dirtier than our compounds," Linsley said. They kept finding the same results, and finally concluded that the siRNA could "cross-react" with other genetic targets. They had trouble convincing reviewers to get their results eventually published in Nature Biotechnology in June 2003. |
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.
Updating and re-interpreting the sacred textAccording to the Central Dogma, DNA, the genetic text, is read out into RNA and RNA is translated into protein. RNA is rather like the scribe copying and translating the sacred text to direct the faithful. But geneticists are now uncovering a vast underworld of heresy to the Central Dogma where RNA agents not only decide which bits of text to copy, which copies get destroyed, which bits to delete and splice together, which copies to be transformed into a totally different message and finally, which resulting message - that may bear little resemblance to the original text - gets translated into protein. RNAs even get to decide which parts of the sacred text to rewrite or corrupt. The whole RNA underworld also resembles an enormous espionage network in which genetic information is stolen, or gets re-routed as it is transmitted, or transformed, corrupted, destroyed, and in some cases, returned to the source file in a totally different form. And this underworld is big, really big. The protein-coding sequence is only about 1.5% of the human genome. Yet, around 97 – 98% of the transcriptional readout of the human genome is non-protein-coding RNA. This estimate is based on the fact that intronic RNA makes up 95% of the primary protein-coding transcripts on average, and there are large numbers of non-coding RNA transcripts which may represent at least half of all transcripts. Most of the miRNAs (microRNA, see below), for example, are derived from (intergenic) regions between genes; and almost half of all transcripts from the mouse genome are non-coding RNAs. A similar estimate applies to the human genome [1]. The inescapable conclusion is that the job of mediating between DNA and protein is really the centre stage of molecular life. And who gives orders to the multitudes of RNA agents? In a sense it is everyone and no one, because the system works by perfect intercommunication. It is not the DNA, but rather, the particular environment in which the RNA agents find themselves. For the organism (organization) to survive, it needs to turnover the DNA text continuously, adapting to the realities of its environment. In the process, it keeps certain texts invariant (see "Are ultra-conserved elements indispensable?" this series), while changing others rapidly in non-random ways (see "To mutate or not to mutate", this series). It also needs to keep referring to texts that are relevant, modifying it, or updating the interpretation in keeping with the times (see "Keeping in concert" this series). |
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 [4] (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,” said Kay.
It had better work, because companies have been testing RNAi on people for treating a respiratory virus and macular degeneration since October 2004.
| RNA interference
(RNAi) was first discovered in
the nematode worm, C. elegans in the
1990s. Researchers noticed that injecting either
sense RNA (the sequence that gets read and
translated into protein) or antisense RNA (the
complementary sequence, which does not code for
protein) into the worm led to specific silencing
of the gene involved. It was later found that the
phenomenon was actually caused by double-stranded
RNA (dsRNA) contaminating the sense or antisense
RNA. RNAi now refers to all gene-silencing
induced by dsRNA. These include a host of other phenomena discovered at around the same time [2, 3]. For example, a gene could be silenced, or ‘co-suppressed’, simply by introducing an extra copy into the genome as a transgene, and transgenes themselves may be silenced either at or after transcription. The coat protein gene of a virus transferred into a plant may protect the plant from the virus, by silencing the virus’ genes. All these phenomena are interlinked through special pathways of RNA processing that are only just being defined (see Fig. 1). Abnormal single stranded RNA (ssRNA) is turned into a double stranded RNA (dsRNA) by an RNA-dependent RNA polymerase enzyme (RDRP). The dsRNA is then chopped up into small pieces or microRNA (miRNA) by the enzyme Dicer. The same enzyme also processes certain hairpin RNA (hpRNA) and related pre-microRNA (pre-miRNA) into miRNA. The miRNA is further processed into single-stranded RNA that’s incorporated into a multiprotein complex called RNA-induced silencing complex (RISC). At this point, the single stranded RNA fragment binds to complementary part of the messenger RNA and either causes the breakdown of the mRNA or prevents its translation into protein. Remember that all this depends on complementary base pairing, just as in DNA, so these mechanisms could potentially exist for each and every one of the now estimated 24 500 genes in the genome. |
“There’s something 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, Ohio, USA
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 up there.
Is the virus to blame? Apparently not, there were no problems injecting ‘empty’ virus without the RNA genes.
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.
Despite these 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 [5].
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.
Toxic shock and leukemiaThe public first became aware of the dangers of gene therapy when healthy teenage Jesse Gelsinger died in September 1999 as the result of volunteering for a clinical trial for the inherited condition Ornithine Transcarbamylase Deficiency. He died of toxic shock after receiving the adenovirus vector carrying the transgene. The ensuing enquiry turned up more than 600 serious adverse events (including deaths) in other gene therapy clinical trials that were unreported, because they were deemed related to the trial procedure ("Failures of gene therapy", SiS 16). Gelsinger’s father sued the research team and subsequently settled out of court for an undisclosed amount. In October and December 2002, the Necker Hospital in Paris announced that the two youngest boys enrolled into a gene therapy study for the treatment of X-SCID had developed a form of leukaemia ("Gene therapy’s first cancer victim", SiS 17). The retroviral vector had inserted near the gene LMO2, which encodes a transcription factor, whose over-expression has been implicated in child-hood T-cell acute lymphoblastic leukaemia.disorders. One child has died. A third infant has developed leukaemia by January 2005, which prompted the US Food and Drug Administration to suspend three gene-therapy trials on SCID in the US. The US Panel has announced that gene therapy for X-linked SCID could proceed only if patients have failed to respond to other treatments. This restriction does not apply to other SCID cases. These two incidents highlight the two major obstacles to gene therapy and the dangers posed: immune reactions against the vectors and transgenes, and inappropriate insertion of vectors and transgenes that can cause mutations leading to cancer. |
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 [6] (Gene therapy woes, SiS 26). Numerous RNA species interfere at every level of gene function [4] (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 [7] (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.
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Find out why the whole biotech enterprise, from GM crops and gene drugs to human cloning, is a phenomenal waste of public finance and scientific imagination, and, most importantly, what it means to be living with the fluid genome.
From the author of the international bestseller, Genetic Engineering Dream or Nightmare? Turning the Tide on the Brave New World of Bad Science and Big Business, 1998, 1999.
- A personal account of one scientist’s struggle against a corrupted scientific establishment bent on promoting genetic modification (GM).
- A dossier of scientific evidence of the most serious GM hazards
- An exposé of the degenerate research programme of mainstream biology
- A death-blow to genetic determinism
- A refreshing antidote to the Darwin industry
Honest * Hard-hitting * Humane
"This enlightening book takes you through the
theoretical and empirical evidence on why we must totally
reject the creation of Frankenstein foods."
Edward Goldsmith, Founding Editor, The Ecologist
"Probably the most disturbing book
of recent times, on a par with Rachel Carson's Silent
Spring. Opens the lid on the intellectual corruption of
Western science."
Noel Lynch, Green Member of the London Assembly, UK
"A trenchant, lucid and thrilling account of how
a multi-billion industry has been built on fraudulent
concepts."
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"An exposé of the risks of genetic engineering
and a warning to developing countries that new
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...........................................
The Author
Dr. Mae-Wan Ho, long-time critic of neo-Darwinism and pioneer of a ‘physics of organisms’, is one of the most influential and widely sought-after speakers in the new paradigm of organic science. As Director and co-founder of the Institute of Science in Society and scientific advisor to the Third World Network, she has had plenty of opportunity to put her science in action.
What this book is about
One of the most persistent dogmas in western science is genetic determinism, the belief that our genetic makeup, or our birth, ultimately determines who and what we are.
Genetic determinism was the guiding principle in the development of the modern science of genetics, which in turn gave rise to the eugenics movement that lasted at least until the mid 1970s in the United States and Europe. Not only were ‘inferior races’ persecuted, ‘inferior’ and ‘disabled’ individuals were also considered ‘unfit’ and targeted for elimination.
Eugenics is surfacing again as human ‘genomic’ science, spawned by the sequencing of the human and other genomes, is promising to identify all the ‘bad’ genes that cause diseases and disabilities, so they could be eliminated at conception or before birth, while the ‘good’ genes would be promulgated, and better yet, used for the ‘genetic enhancement’ of anyone who can pay for the privilege. This time round, eugenics will not be sanctioned by the state. It will be up to the ‘global market’ to decide. The poor will become a genetic ‘underclass’. Social inequality will be redefined as, and transformed into genetic inequality.
Fortunately, science as knowledge of nature is never just subject to our arbitrary whim and prejudice. We can delude ourselves, but only for so long. Nature has a way of fighting back, of puncturing our illusions.
The story of the ‘fluid genome’ tells how geneticists came face to face with scientific findings that completely undercut the old genetic determinist paradigm. It makes nonsense of all the eugenicist claims and promises, and exposes the futility as well as the hazards of genetic engineering for the health of human beings and the entire life-supporting system that’s our planet.
Read this book to find out what it means to be liberated from the genetic determinist myth and to be living with the fluid genome.
A few quotations from the book:
"This [neo-Darwinian] theory is very comforting for those who derive the most benefit from the status quo, and they are responsible for a huge ‘Darwin industry’ dedicated to ‘explaining’ why this is the best of all possible worlds, which is masquerading as mainstream science."
"An ‘academic-industrial-military complex’ has matured with the rise of gene biotechnology that’s increasingly active in suppressing scientific dissent in the genetic engineering debate, threatening the survival of science and endangering lives."
"Genes and genomes are part and parcel of the entire physiological system that responds to the environment in myriad non-random, repeatable ways….It is ironic that Lamarckian principles should now be found to be operating at the molecular level."
"Doing science is rather like solving a murder mystery. You have to follow your hunches, which may often get you into blind alleys, you have to watch out for clues, interpret the evidence from experimental findings and connect them up into a coherent whole. And then, you have to present the case to your peers and the public, as though you are in a court of law."
"Genetic engineering greatly enhances horizontal gene transfer and recombination, the very processes that create new viruses and bacteria that cause outbreaks of infectious diseases and spread drug and antibiotic resistance."
"The responsiveness of genes and genomes to the environment makes clear that the only way to keep genes and genomes constant and healthy is to have a balanced ecology..… On the other hand, it is definitely futile to think that we can go on ruining our ecosystem and stay healthy so long as we have ‘good’ genes…Genes, unlike diamonds, are not forever."
"How molecules and cells can work together in the body is ultimately by intercommunication and reciprocity. In the ideal of quantum coherence, each gene and protein is as much in control as it is sensitive and responsive."
"What does quantum coherence look like in the body? Perfectly fluid, dynamic and scintillating, which is why the genome is fluid. Think of the diverse activities in the body as ‘quantum jazz’, where every single player, however small, is improvising freely from moment to moment, and yet keeping in beat and in sync with the whole. There is no composer or conductor, the music emerges spontaneously as it is played, in endless variations that never exactly repeat."
"The ideal organic whole, quintessentially pluralistic and diverse, works by total participation and intercommunication, and is at once most coherent and most free, for the parts as for the whole."
200pp £7.99 (£10 inc. postage worldwide)
Publication date: 30 April 2003
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