Ensuring the safe use of genetic engineering is much more important than preventing or defending the nation from bioterrorist attacks Dr. Mae-Wan Ho
'Biosecurity' originated from a small group of scientists who met in 2001 to discuss how to keep diseases affecting crops and livestock from crossing national boundaries . Then, came the anthrax attacks post September 11, and the term came to be used for measures aimed at countering terrorist attacks involving biological agents or toxins. Suddenly, thousands of US scientists are caught in a web of new rules for keeping dangerous agents and substances, and even scientific knowledge, out of reach of bioterrorists. Biosecurity should come under the Biological Weapons Convention (BWC) to which the US is signatory; but the US has rejected the Convention's remit to establish a procedure to verify compliance with the Convention ("Bioweapons Convention -no progress in sight", SiS 13/14) .
'Biosafety' refers to a set of measures aimed at regulating and ensuring the safe use of genetic engineering and transnational movements of genetically modified organisms. It falls within the scope of the Cartagena Biosafety Protocol under the Convention of Biological Diversity. The US is not a party to the Biosafety Protocol and has steadfastly refused to acknowledge it. The US position is that genetic engineering biotechnology is inherently safe, and only its misuse needs to be prevented.
It is clear that the BWC and Cartagena Biosafety Protocol overlap, and are both needed for effective control of genetic engineering and biological weapons. Of the two, biosafety is the more critical, although most of the attention is focussed on biosecurity.
A report from the National Academy of Sciences , Biotechnology Research in an Age of Terrorism: Confronting the Dual Use Dilemma, grew out of a meeting in January 2003 chaired by Gerald Fink, Professor of Genetics at the Whitehead Institute for Biomedical Research at Massachusetts Institute of Technology.
The Report recommends a review process both at the research stage and at the stage of publication. Its stated aim was to "safeguard the integrity of science", and not "to censor research or research publications". It proposes "a system of filters" to help determine whether a particular study should be done, and if so, how the finding might best be published to avoid potential misuse. It emphasizes "voluntary self-governance by the scientific community". The editors of Cell, Science, Nature and PNAS have already agreed to review and "filter" out "sensitive" papers or information.
The Bush administration has adopted the Fink Report, but its implementation is mired in difficulty. The system relies on Institutional Biosafety Committees that frequently fail to comply with federal rules and on a National Science Advisory Board on Biosecurity announced a year ago, but has yet no members .
The US has been caught in a dilemma of its own making because the government has been pouring billions into 'biodefence' research on biological weapons agents. It sets up some 40 new and upgraded 'hot zones' across the country  with hundreds of biosafety level 3 or 4 labs, designed to research the most dangerous pathogens such as Ebola, plus at least 11 doing classified, secretive research, 6 major aerosol facilities and 3 open-air testing facilities. These labs and facilities include not just established bio-weapons research institutions, but also former nuclear weapons installations and top universities, like Boston, Harvard, John Hopkins, California, Illinois, Texas and New York; many situated in the middle of densely populated areas, or in some cases, deliberately sited in remote regions where they can operate in secrecy (e.g., Dugway Proving Ground, Utah or Rocky Mountain Labs, Montana).
Critics call attention to accidental releases of dangerous pathogens and failures of biosafety containment that could spell disaster for residents ("Bio-defence mania grips United States", and "Biodefence contravenes biosafety", SiS 19) . It is difficult to distinguish bio-defence research from bio-weapons research. In order to make vaccines against deadly biological agents, the deadly biological agents have to be created. The US programme, particularly in the Departments of Defense and Homeland Security, is increasingly focussed on "threat assessment" studies where researchers deliberately create the "threat", i.e., the weapon, claiming they are learning to defend against it.
There is also the danger that researchers will be trained in precisely the techniques needed for bio-terrorism. The post-September 11 anthrax attacks have been traced to the federal bio-defence lab in Fort Detrick Maryland.
Under the newly formed Department of Homeland Security (DHS), much defence-related research and development will be exempt from the Freedom of Information Act, and hence there will be little or no mandatory public disclosure.
Nevertheless, the Sunshine Project (www.sunshine-project.org), a bioweapons watchdog, has developed and released a new webtool (CRISPER) to search and organize research grant data from the National Institutes of Health (NIH). It revealed that the decision of NIH National Institute for Allergy and Infectious Diseases (NIAID) to give priority to research of high biodenfence but low public health significance resulted in 1500% increase in the number of grants awarded for biological weapons agents, while the number of grants for model microorganisms and non-bioweapons pathogens decreased by 41% and 27% respectively.
More than 750 prominent scientists signed an open letter, published in Science, to the director of NIH to express their concern . Richard Ebright of Rutgers University, who initiated the letter, points out that prioritising research on poorly known agents could backfire, not least because of the need for strict containment and new experimental tools. The biodefence money would be better spent researching related, but less pathogenic organisms. He also believes that increasing the number of labs and people working on bioterror agents would increase the risk of an accidental release or deliberate attack.
NIAID's biodefence budget shot up from $42m in 2001 to $1.5bn in 2004, with $1.6 projected for 2005. The government wide total spending on biodefence before 2001 was less than $1 billion/year; between 2001 and 2005, the total spending comes to $22 billion, and the projected total spending between 2001 and 2006 is $30 billion.
One should not underestimate the increased possibilities of creating powerful biological weapons in the post-genomics era ("GM & bioweapons in the post-genomics era", SiS15)  (see Box 1). But that's precisely also why there can be no effective biodefence ("Biodefence in tatter", SiS 15) (see Box 2). The agents are unknown and unpredictable. They can target the immune system directly to undermine the body's defence. Vaccines will be ineffective, or worse than useless as the smallpox vaccine may prove to be; and there is no way to adequately test for efficacy or safety. Theoretical studies indicate that partially effective vaccines may increase the virulence of pathogens. There is no known defence against agents that target buildings or structures.
Post genomics possibilities for bioweapons
Futility of biodefence
The dangers of biodefence were highlighted in January 2005 when three lab workers in Boston University were reported to have fallen ill from being infected with tularaemia . The infections happened between May and September 2004, but did not become public knowledge until a week after Boston's Zoning Commission approved the construction of a biosafety level 4 laboratory in the University.
The workers had handled a live strain of the tularemia bacterium instead of the non-infectious one typically used. They were trying to find a vaccine for 'rabbit fever'.
Before that, there were three lab accidents involving the SARS virus. The Washington Post (29 May 2004) commented : "Scientists still do not fully understand exactly where or how SARS emerged 18 months ago. But it is now clear that the most threatening source of the deadly virus today may be places they know intimately - their own laboratories."
The three lab outbreaks of the disease since September 2003 - in Singapore, Taiwan, followed by the 9 cases linked to China's National Institute of Virology - have wider implications. They "highlighted the unique hazards to public health that arise from accidental releases of germs that no longer exist - or barely exist - in the wild."
The article described the notorious release of smallpox in Birmingham, England, in August 1978, 10 months after the last infection occurred in Somalia.
Henry S. Bedson, head of the microbiology department at a medical school, was rushing to finish his experiments before the deadline to turn in or destroy his stocks of smallpox, as his lab had been judged unsatisfactory by the World Health Organisation inspectors. The smallpox virus apparently became airborne, and transported up one floor through air ducts to a photographic studio and darkroom to infect a 40-year-old photographer who died, even though she had been vaccinated 12 years earlier. But not before she transmitted the virus to her mother, who also became ill but survived. Her father did not become infected but died from a heart attack. Bedson slashed his throat, leaving a note that said, "I am sorry to have misplaced the trust which so many of my friends have placed in me and my work."
The Council for Responsible Genetics  listed numerous breaches of bio-containment of disease agents in the US: involving accidental and deliberate environmental releases, failures of containment, loss of samples and 13 cases of exposures and infections of personnel between 1994 and 2004. The agents included AIDS, Ebola virus, West Nile virus, glanders, plague, anthrax and tularemia.
I have stressed that the control of bioweapons and genetic engineering must go together (SiS 13/14) , and that the hazards from genetic engineering could be worse than bioweapons : The basic tools and materials for making bioweapons are the same as those used in 'legitimate' genetic engineering applications. But while bio-weapons are made under strictly contained conditions, many dangerous experiments are being done without adequate safety precautions, and hazardous gene products are released into the environment as if they were safe.
More and more scientists are voicing concerns over genetically modified crops (The Case for a GM-free Sustainable World and "ISP letter to FDA" ) [15, 16]: the potential toxicity and allergenicity of transgenic products and their negative impacts on biodiversity, as well as the horizontal transfer of transgenic DNA and antibiotic resistance marker genes. The more serious and insidious hazards, however, are associated with contained uses of genetic engineering biotechnology.
The Fink Report  dismissed the risks of genetic engineering: "The initial fears about the inadvertent creation of virulent microbes by gene splicing techniques have abated because of overwhelming scientific evidence to the contrary. There have been no reported cases of disease caused by recombinant microorganisms despite the widespread use of gene splicing techniques in academic laboratories and in the production of pharmaceuticals."
I co-authored a paper with six scientists entitled, "Gene technology and gene ecology of infectious diseases", published in 1998 , summarising all the evidence suggesting that genetic engineering may have contributed to the resurgence of infectious diseases since commercial scale genetic engineering began; calling for an independent public enquiry.
I sent a preprint to the World Health Organisation and the UK Health and Safety Executive. Eventually, the HSE wrote and said it would commission its own review, and nothing more was heard. But the question we raised in the paper remains as alive as ever ("SARS virus genetically engineers?" SiS 19) .
The Fink Committeee identified seven kinds of "experiments of concern" as those having "a greater likelihood for potential misuse", and hence would be subject to further examination (Box 3).
Experiments of concern from Fink Report 
Practically all seven classes of experiments are being done in genetic engineering, in the course of genetic modification of bacteria, plants and animals or human beings in 'gene therapy'.
Antibiotic resistance marker genes have been released into the environment with GM crops in field trials or for commercial growing. Bacteria and viruses are routinely mutated and recombined in the laboratory, which could change their host range, increase their virulence or transmissibility or render existing vaccines ineffective. In fact, simply strip off
the coat from a virus, and the naked genome would be taken up by non-host cells to generate infectious viruses . Researchers have been culturing human viruses in animal cells for decades, which would certainly alter their host range. They also culture viruses in mixtures of cells from different species deliberately to obtain mutants that infect both species, the latest involving the SARS virus . Similarly, gene therapists have been wrapping gene transfer vectors in liposomes and other material to escape immune detection, which is a major problem with gene therapy vectors . These vectors, though disarmed, have the potential to regenerate live viruses or to cause insertion mutagenesis. A third case of leukemia has surfaced among the children given gene therapy for X-linked SCID in Paris . Finally, almost any bacterium can be 'weaponised' by transferring into it whole sets of virulence genes in mobile 'pathogenicity islands' or phage coding for toxins ; in experiments that are routinely done in genetic engineering.
A research team in the State University of New York at Stony Brook made the poliovirus by joining up chemically synthesized short DNA fragments into a complete sequence that was then transcribed into the RNA viruses in a cell-free system containing all the necessary enzymes and cell parts . The synthetic viruses were capable of infecting cells.
The researchers demonstrated that one could synthesize any virus from chemical reagents that can be purchased in the open market, using the public database for the genome sequence. The experiment is not exactly new. David Baltimore and colleague  had shown in 1981 that a DNA copy of the RNA genome of poliovirus could be taken up into living cells to generate infectious virus.
Also in 2002, researchers in the University of Pennsylvania, Philadelphia, showed  that a gene in the smallpox variola virus is more than 100 times more potent than the version in vaccinia virus (used in vaccines against smallpox) in inhibiting the human complement enzymes protecting the body innate against viral attacks. And that could be why the smallpox virus is so much more virulent than the vaccinia virus.
The gene from the variola virus, therefore, has the potential to increase the transmissibility of other viruses; but, as claimed, disabling it may be "therapeutically useful if smallpox re-emerges".
Should scientists do this kind of experiments and report the results openly? One could argue that open knowledge is always a good thing, because it alerts people to the possibilities and encourages them to be vigilant and to find means of overcoming the dangers. Whether they should do the experiment in the first place is questionable.
The US is currently proposing to express variola genes in related pox viruses, and to insert a reporter gene (expressing green fluorescent protein) in variola itself . The World Health Assembly is to consider these controversial proposals in May 2005.
AIDS researchers have pointed out that the gp120 protein is strongly immunogenic, but the antibodies fail to protect against the virus. Instead, it ends up over-stimulating the immune system, leaving it less able to cope with new infections. Moreover, the part of the gp120 molecule that plays the dominant role in provoking an immune response is the V3 loop. The V3 loop and flanking regions are similar in base sequence and structure to the antigen-binding region of the human immunoglobulin (Ig). It has been suggested since the early 1990s that this immunoglobulin-like domain in gp120 may interfere with the immune regulatory network. The V3 loop and its flanking regions are, moreover, located between recombination signals similar to those found in human immunoglobulins, and to the Chi recombination hotpots found in many viruses and bacteria. Consequently, the immunologically dominant region of gp120 may be involved in recombining with human immunoglobulin genes resulting in autoimmune responses, and may also recombine with co-infecting viruses and bacteria to generate new pathogens. Evidence of recombination between gp120 and bacterial DNA has subsequently been found in the sera of AIDS patients.
The gp120 gene, spliced into numerous bacterial and viral vectors with Chi or Chi -like sequences as vaccines, therefore multiply the odds for recombination to generate new pathogens. The gp120 gene has been incorporated into GM maize as a cheap edible vaccine against HIV; while the key bioweapons institute in Russia has created an artificial protein consisting of the env and gag antigens from HIV fused with the hepatitis B virus protein HbsAg as a HIV/hepatitis B combined vaccine. This is tantamount to releasing slow bioweapons on the populations. The new combinations of dangerous genes will have plenty of opportunity for generating new pathogens through further gene trafficking with bacteria and viruses in the environment, of which less than 1% can be cultured and identified.
Also in connection with AIDS, researchers have created a hybrid simian and human immunodeficiency virus (SIV +HIV = 'SHIV') for testing vaccines, which kills victim primates in weeks.
Some of us have been warning that genetic engineering is inherently dangerous because it greatly multiplies the scope and frequency of horizontal gene transfer and recombination, the major route for creating new viruses and bacteria that cause disease epidemics. This was brought home when researchers in Australia 'accidentally' transformed a harmless mousepox virus into a lethal pathogen that killed all the mice, even those that were supposed to be resistant to the virus .
Headlines in the New Scientist editorial : "The Genie is out, Biotech has just sprung a nasty surprise. Next time, it could be catastrophic." The lead article continued in the same vein : "Disaster in the making. An engineered mouse virus leaves us one step away from the ultimate bioweapon."
The researchers added a gene coding for the immune signalling molecule interleukin-4 to the virus, which they thought would boost antibody production; instead, it suppressed both primary antiviral immune responses as well as adaptive immune responses. The same gene, spliced into a vaccinia virus previously, delayed the clearance of virus from the animals; so it may well have the same immune suppressive effects for all viruses.
More surprisingly, a paper published in December 2003 described how disrupting a set of virulence genes in Mycobacterium tuberculosis, the tuberculosis bacterium, resulted in a hypervirulent mutant strain that killed all the mice by 41 weeks, while all the control mice exposed to the wild-type strain survived .
These two cases underscore the complexity of disease generation by pathogens. They also leave us in no doubt that apparently innocent experiments could end in nasty surprises.
There have been previous cases that went unnoticed. A paper published in 2001  described how a method established for recovering infectious Ebola virus by reverse transcription enabled the researchers to produce a mutant considerably more toxic to the host cells than the wild-type.
Finally, Willem Stemmer of Maxygen Inc. based in California invented a DNA shuffling technique in which genes, viral and bacterial genomes, whatever, could be chopped up into fragments, then made to join up at random into millions of recombinants, out of which superior performing enzymes, metabolically more efficient genomes or more infectious and virulent pathogens could all be selected .
In one experiment, 6 mouse leukemia viruses (MLVs) were recombined in a single round giving 5 million replicating recombinant viruses in a matter of hours. Among them were completely new viruses that infected Chinese Hamster Ovary cells, which none of the original MLV was capable of. Some recombinants were 30 to 100 times more stable than the parental strains. There is no way to characterize all of the 5 million recombinants and they may well include new killer viruses.
Researchers have given up trying to cope with the massive complexity of the genome and gene function ("Life after the Central Dogma" series, SiS 24) . Instead of rational design, they have resorted to random genome shuffling to improve industrial microbes , and the advantage is that the resultant microbes aren't even classified as genetically engineered, and therefore not subject to usual regulation and can even be used in the food industry, we are told. They even suggest using environmental libraries, containing the DNA of the 99% bacteria unknown to science, in genome shuffling; but are at least aware of the dangers involved: "New drug resistances" and emergence of "accelerated or new pathogenic mechanisms or diseases".
It has been known for some time that bacterial and viral DNA can cause immune reactions, because that's part of our innate immune response that protects us from germs; and this has become a major obstacle to gene therapy . Any fragment of double-stranded DNA or RNA down to 25bp is immunogenic. There is now new evidence that certain sequences of single stranded RNA also elicit specific immune reactions .
Biotechnological processes and genetic engineering labs are creating an increasing variety of naked/free nucleic acids that are currently released unregulated into the environment, where they can elicit immune reactions, as well as undergo uncontrolled horizontal gene transfer and recombination to generate new pathogens or trigger cancer, should some of them jump into the genome of our cells.
Compare the list of "Dangerous experiments constructs and deliberate releases in genetic engineering" (Box 4) with the "Experiments of concern" in the Fink Report (Box 3). The list for genetic engineering is more extensive and more insidious; as the deadly biological agents generated cannot be predicted, nor the millions of cancers that may result from insertion of constructs with strong viral promoters.
Dangerous experiments, constructs and deliberate releases in genetic engineering
The good news is that practically all the hazards of genetic engineering can be prevented: by tightening the regulation of contained use as has been done for deliberate release. I wrote a report with three colleagues in 2001 , Slipping through the Regulatory Net: 'Naked' and 'Free' Nucleic Acids. The report was reprinted 2002 and 2004, because our message has not yet got through to the regulators.
Current European regulation allows users to release directly into the environment certain live transgenic microorganisms considered nonpathogenic or otherwise safe in liquid waste, although there is no agreement across European countries as to which bacteria are pathogens.
Meanwhile, all killed microorganisms and cells containing transgenic DNA are disposed of as solid waste, and are either recycled as food, feed and fertilizer, or disposed of in landfill.
There is an urgent need for validated procedures to ensure that all kinds of transgenic nucleic acids from contained use are thoroughly degraded before transgenic wastes are released into the environment.
Meanwhile, there should be no environmental releases of GM crops unless and until they can be proven safe beyond reasonable doubt.
Article first published 16/03/05
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