Last Updated on 4/11/00
By Rachel C. Benbrook
Email: karen@biotech-info.net
Mae-Wan Ho and Angela Ryan* It has long been our contention that genetic engineering is inherently hazardous, if only because of the rampant, arbitrary combinations of genetic material it creates. The most dangerous category of recombinant genetic material are perhaps the artificial vectors for carrying and transferring genes, which are generally made of viruses that cause diseases and other genetic parasites that spread virulence and drug and antibiotic resistance. Unlike natural viruses and genetic parasites, which respect species barriers, artificial vectors are designed to be promiscuous, and to be able to jump ultimately across all species barriers. These are also designed for invasiveness, to overcome mechanisms that guard against their integration and expression in foreign genomes. In a recent ISIS report (1) produced for circulation at the Biosafety Protocol Meeting in Montreal (Jan. 2000), we pointed out that an increasing variety of naked/free nucleic acids are now being made for use in research, industrial productions and medical applications, all of which are being released unregulated into the environment. They range from oligonucleotides to artificial constructs thousands and millions of basepairs in length, often containing heterogeneous arrays of genes from pathogenic bacteria, viruses and other genetic parasites belonging to every kingdom of living organisms. As most of them have never existed, or if they have, not in such large amounts, they are, by definition, xenobiotics - substances foreign to nature - with the potential to cause harm. Some gene therapy vectors and vaccines have already been found to elicit toxic and other harmful reactions. Nucleic acids are now known to persist in all environments, including the digestive tract. Transformation by the uptake of DNA is a significant route of horizontal gene transfer, and there is overwhelming evidence that horizontal gene transfer and recombination have been responsible for the recent resurgence of drug and antibiotic resistant infectious diseases. Recent research in gene therapy and in vaccine development leaves little doubt that naked/free nucleic acids are readily taken up by the cells of all species of mammals including human beings, and may become integrated into the cell's genome. Integration can result in harmful biological effects, including cancers. There is also the potential for generating new viruses by horizontal gene transfer and recombination. In short, the need to establish regulatory oversight of such artificial constructs at both national and international levels is long overdue. We report on several new categories of constructs below: powerful synthetic promoters, hybrid gene-therapy vectors that combine high infectivity with the ability to integrate into genomes, a vector made from an insect virus with mammalian viral promoters that can infect practically all mammalian cells at high frequency, and synthetic jumping genes that can infect bacteria as well as all higher organisms. Promoters are gene switches for turning genes on, and every functioning gene has to have one. Promoters typically have a modular construction, consisting of parts that respond to different signals from a battery of other genes, which determine where and when they are turned on, by how much and for how long. Thus, promoters allow genes to talk to one another. These gene-conversations form complex intercommunication networks that enable the tens of thousands of genes in an organism to function as a coherent whole, and to respond appropriately to the environment. Researchers in Texas are developing synthetic superpromoters that surpass the capacity of natural promoters to boost gene expression (2). The scientists took four different regulatory elements from muscle-specific promoters and reassembled them at random to make new synthetic promoters. Some of the resulting synthetic promoters give gene-expression levels greatly in excess of those of normal muscle promoters or viral promoters. The promoter elements used were:
What will these synthetic promoters do to gene-regulation in the organism as a whole? This question is almost never asked by genetic engineers whose focus is entirely on the function of the one or two transgenes controlled by the promoters. As every regulatory element of a promoter is embedded in an intricate web of highly specific modulations of gene functions, simply recombining the elements will have unintended global (pleiotropic) effects. When the resultant promoter makes genes over-express as well, there could be major disruptions to the genetic intercommunication networks. What are the potential hazards? Again, this question is seldom considered. The recombination of synthetic superpromoters with cellular proto-oncogenes may give rise to cancer on account of over-expression of the proto-oncogenes. Similarly, recombination of the superpromoters with dormant viruses and other infectious genetic elements in the genome may create infectious viruses and other invasive elements. It is already known that the CAT motif is also active in SV40, a virus now found to be associated with many human cancers. Gene therapy is fraught with difficulties and pitfalls (see ISIS Report, ref. 1) Gene therapy vectors (and naked DNA vaccines) have caused acute toxic shock reactions and severe immune reactions. Between 1998 and 1999, there were six deaths and more than 650 adverse events resulting from clinical trials of gene therapy, the causes of which are yet to be determined. Naked DNA can also trigger autoimmune reactions. Recent research indicates that any fragment of double-stranded DNA or RNA (down to 25 base-pairs) introduced into cells can induce autoimmune reactions, which are linked to rheumatoid arthritis, insulin-dependent diabetes and Graves disease of the thyroid (see ISIS News #2). Many 'spontaneous' mutations result from insertions of transposons and other invasive elements into genes. Insertion mutagenesis is associated with a range of cancers of the lung, breast, colon and liver. Gene therapists are finding it almost impossible to get gene therapy vectors efficiently and safely delivered into target tissues and to produce therapeutic amounts of gene product for an appropriate time. Now, dangerous recombinant viral vectors are under development in a desperate attempt to overcome the problems. Researchers have created an adenovirus-retrovirus vector by combining the long terminal repeats (LTRs) of the Moloney murine leukemia retrovirus - which has the ability to integrate into genomic DNA, with adenoviral vectors - which have high infection efficiency (3). An integration frequency of 15% was observed and expression persisted for nine weeks (the last time point studied), being 15 fold greater in the hybrid vector than in the control. This work demonstrates that adenoviral vectors containing retroviral LTR elements are able to integrate in the absence of the retroviral structural proteins, which were previously thought to be necessary. The scientists point out that a large variety of retroviral sequences exist within genomes and are known to be present in mammals including humans. These elements may have provided helper-functions to the hybrid vector. Random integration was observed and sequence analysis found no site-specific target sequence. Insertion occurred over different chromosomes, and multiple insertions were seen within individual cells. Many unproductive integrations were also found as the result of the vector breaking, a sign of structural instability. In an attempt to achieve more efficient gene transfer and expression in vivo the researchers have, in effect, created the potential for a new supervirus that will have the infectivity of the adenovirus and the ability of the retrovirus to invade genomes. Baculovirus, previously thought to be specific for insect cells and widely manipulated for controlling insect pests in agriculture, was recently found to infect mammalian cells. It is therefore being actively developed as a vector for gene therapy. We have pointed out the hazard, obvious to us, though not apparently to the genetic engineering community, that genetic engineered baculoviruses used in agriculture may infect agricultural workers, food processors as well as the public at large (4). Researchers at the Glaxo Wellcome Research Institute have created a recombinant baculoviral vector intended for gene therapy containing two mammalian gene-expression cassettes with constitutive promoters. (A constitutive promoter is one that gives continuous gene expression.) The cytomegalovirus (CMV) immediate early promoter/enhancer is used with the green fluorescent protein gene, and the simian virus 40 (SV40) early promoter is used with the antibiotic resistance marker gene for neomycin phosphotransferase II (NPTII). The antibiotic Geneticin is used to select for cells that have taken up the recombinant baculoviral vector. High frequencies of uptake (72% to 94%) were found for a variety of mammalian cell lines: those originating from human hepatomas, from pig kidney and from a variety of other tumours of pig, rat and human. From these cells that have taken up the vector, further lines could be selected that express the genes stably, suggesting that the vector may have integrated into the cells' genome. The vector is probably promiscuous for all mammalian cells as well as insect cells. It has the potential to generate new viruses that cross from insects to mammals and vice versa. Investigative journalist Edward Hooper has written a book detailing the circumstantial evidence that the AIDS virus, HIV may have been created in the manufacture of polio vaccines in monkey cells (5). And researchers have pointed out that this should give grounds for caution in xenotransplantation (6). In our view, gene therapy vectors are even more likely to facilitate the evolution of cross-species viruses. David Baltimore, Nobel laureate and president of Caltech who works in gene therapy is now coming out fervently against its practice. He said recently in an interview "I disagree we've had value from gene therapy trials so far. A number of us are asking, 'what the hell are we doing putting these things into people?' ". We strongly share his views. Finally, scientists in Boston have developed a new breed of transposons or jumping genes as "a universal tool for genetic studies in bacteria" (7). The construct is derived from mariner, a superfamily of transposable elements found across genomes of diverse eukaryotic organisms. The mariner family element - Hirmar1 , isolated from the horn fly was used to make this new transposition system, named 'minitransposons'. It employs the short inverted repeats of Hirmar1 flanking a kanamycin antibiotic resistance marker gene that is driven by a bacterial promoter. Transposition and random insertion in E. coli and in Mycobacterium was observed, with little site-specificity beyond the known requirement for the dinucleotide TA. Within the 500 base pairs analysed, insertion was shown in 21 of the 23 possible TA dinucleotide insertion sites. Significantly, the scientists found one insertion that caused the activation and over expression of a host gene. The insertion site was 164 basepairs away from the gene; the bacterial promoter used to drive the expression of the antibiotic resistance gene had inserted in the same orientation and was therefore able to drive its over expression. The authors report the system as "universal", which means it can operate in any organism and they suggest it should be widely used to study many important pathogens. Considering the difficulty in maintaining sterile conditions in laboratories, the widespread use of this system poses a serious health risks. It is designed specifically to overcome all species barriers and to insert into all genomes, where it can cause insertion mutagenesis. There must be stringent measures to prevent these and other artificial constructs from being released into the environment in any form. Nor should they be used for gene therapy or to create transgenic organisms that are released into the environment. Civil society should be debating whether such hazardous research of no obvious benefit should be supported. References
Open University Walton Hall, Milton Keynes MK7 6AA, UK m.w.ho@open .ac.uk I-sis@dircon.co.uk ** NOTICE: In accordance with Title 17 U.S.C. Section 107, this material is distributed for research and educational purposes only. **
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Last Updated on 4/11/00 By Rachel C. Benbrook Email: karen@biotech-info.net |
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