ISIS Report -Produced for the Third World Network
Mae-Wan Ho, Angela Ryan; Biology Department, Open University, UK
J.Cummins; Department of Plant Sciences, University of Western Ontario, Canada
T. Traavik; Dept. of Virology, Institute of Medical Biology, Norway
A huge variety of naked/free nucleic acids are being produced in the laboratory and released unregulated into the environment. They are used as research tools, in industrial productions and in medical applications such as gene therapy and vaccines. These nucleic acids range from oligonucleotides consisting of less than 20 nucleotides to artificial constructs thousands or millions of basepairs in length, typically containing a heterogeneous collection of genes from pathogenic bacteria, viruses and other genetic parasites belonging to practically every kingdom of living organisms. Most of the nucleic acids and constructs have either never existed in nature, 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, such as gene therapy vectors and vaccines, have already been shown to elicit toxic and other harmful reactions in preclinical trials.
Nucleic acids are now known to persist in all environments, including the digestive system of animals. Transformation by the uptake of DNA is recognized to be a significant route of horizontal gene transfer among bacteria, 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 investigations associated with gene therapy and vaccines leave little doubt that naked and free nucleic acids are readily taken up by the cells of all species including human beings, and may become integrated into the cells genetic material. There is also abundant evidence that the extraneous nucleic acids taken up can have significant and harmful biological effects including cancers in mammals.
The need to establish regulatory oversight of naked/free nucleic acids at both national and international levels is long overdue. It is irresponsible to continue to exclude them from the scope of the International Biosafety Protocol.
Naked nucleic acids are DNA/RNA produced in the laboratory and intended for use in, or as the result of genetic engineering (1).Free nucleic acids refer to the laboratory-produced nucleic acids transfected into cells or organisms, whether incorporated as transgenic DNA or not, and subsequently released into the environment by secretion, excretion, waste disposal, death, industrial processing, or carried by liquid streams, or in airborne dust and pollen.
A huge variety of naked nucleic acids are being produced in the laboratory (see Box 1), which are used as research tools, in industrial productions, and in medical applications such as gene therapy and vaccines. They range from oligonucleotides consisting of less than 20 nucleotides to artificial constructs thousands of basepairs in length, and artificial chromosomes millions of basepairs long. The constructs typically contain antibiotic resistance marker genes plus a heterogeneous array of genes from pathogenic bacteria, viruses and other genetic parasites belonging to practically every kingdom of living organisms on earth (2).Most of the naked nucleic acids and constructs have either never existed in nature, or if they have, not in such large amounts. They are, by definition, xenobiotics substances foreign to nature -(3) with the potential to cause harm.
There is no regulation governing the release of naked nucleic acids into the environment. Many novel constructs are incorporated into transgenic micro-organisms and animal cell cultures for commercial drug production, and into crops, livestock, fish and other aquatic organisms for food, animal feed, and other purposes. These constructs are therefore greatly amplified, and at the same time introduced into foreign genomes where recombination with host genes and the genes of the hosts viral pathogens may readily occur. Transgenic wastes containing large amounts of free or potentially free transgenic DNA are being released unregulated into the environment, including those from microorganisms and cell cultures supposed to be in contained use (see Box 2)(4).Under the current EU Directive for Contained Use, contained users are allowed to release certain live transgenic microorganisms in liquid waste, and all killed microorganisms and cells containing transgenic DNA as solid waste.
Naked nucleic acids in genetic engineering biotechnology
DNA - based
Viral genomes, eg, cauliflower mosaic virus, cytomegalovirus, vaccinia, baculovirus, adenovirus, SV40, many bacteriophages
cDNA of RNA viral genomes, eg, retroviruses SIV, HIV, Rous Sarcoma virus, mouse Moloney virus
Plasmids, eg, Ti of Agrobacterium, many plasmids from E. coli and yeast, often carrying antibiotic resistance genes
Transposons, eg, many broad host range transposons from E. coli with antibiotic resistance genes, some from Drosophila, such as mariner are found in all kingdoms
Artificial vectors made by recombining viral genomes, plasmids and transposons, carrying one or more antibiotic resistance genes; used for gene amplification, DNA sequencing, transfection, gene therapy, etc(5)., many are shuttle-vectors designed for replication in more than one species, pantropic vectors cross many species barriers
Naked DNA vaccines, plasmid-based, viral vector based(6)
Artificial chromosomes: yeast (YAC) plasmid (PAC) and mammalian (MAC) made from telomeric and centromeric repeat sequences(7)
Artificial constructs: transgene cassettes, often include antibiotic resistance gene cassettes
PCR amplified sequences
Oligodeoxynucleotides (antisense), hairpin-forming oligodeoxynucleotides used in gene therapy(8)
RNA - based
Antisence RNA used in gene therapy
Ribozymes used in gene therapy(9)
Self-replicating RNA (linked to RNA-dependent RNA polymerase) used in gene therapy(10)
Chimeroplasty hairpins used in targeted gene mutation(11)
The lack of regulation of naked/free nucleic acids is based largely on the assumption, now proven to be erroneous, that naked/free nucleic acids would be rapidly broken down in the environment and in the digestive system of animals(12). Another assumption is that as DNA is present in all organisms, it is not a hazardous chemical, and hence there is no need to regulate it as such(13).
Free nucleic acids resulting from genetic engineering biotechnology
Transfected, unincorporated nucleic acids/constructs due to gene-therapy, vaccination, transgenesis, which are released into the environment by secretion, excretion, waste carcass disposal, cell death, etc.
Transgenic DNA released from live or dead cells contained in:
Naked or free DNA are now known to persist in all natural environments, and high concentrations are found in the soil, in marine and fresh water sediments as well as in the air-water interface, where it retains the ability to transform microorganisms(14). DNA also persists in the mouth(15) and the digestive tract of mammals(16), where it may be taken up and incorporated by the resident microbes, and by the cells of the mammalian host.
A genetically engineered plasmid was found to have a 6 to 25% survival after 60 min. of exposure to human saliva. The partially degraded plasmid DNA was capable of transforming Streptococcus gordonii, one of the bacteria that normally live in the human mouth and pharynx. Human saliva contains factors that promote competence of resident bacteria to become transformed by DNA(17).
It has long been assumed that DNA cannot be taken up through intact skin, surface wounds, or the intestinal tract, or that it would be rapidly destroyed if taken up. Those assumptions have been overtaken by empirical findings. The ability of naked DNA to penetrate intact skin has been known at least since 1990. Cancer researchers found that within weeks of applying the cloned DNA of a human oncogene to the skin on the back of mice, tumours developed in endothelial cells lining the blood vessel and lymph nodes(18).
Viral DNA fed to mice is found to reach white blood cells, spleen and liver cells via the intestinal wall, to become incorporated into the mouse cell genome(19). When fed to pregnant mice, the viral DNA ends up in cells of the fetuses and the new born animals, suggesting that it has gone through the placenta as well(20). The authors remark that "The consequences of foreign DNA uptake for mutagenesis and oncogenesis have not yet been investigated."(21)
Recent developments in gene therapy demonstrate how readily naked nucleic acids (see Table 2) can gain access to practically every type of human cells and cells of model mammals. Naked nucleic acids can be successfully delivered, either alone or in complex with liposomes and other carriers, in aerosols via the respiratory tract(22), by topical application to the eye(23), to the inner ear(24), via hair follicles(25), direct injection into muscle(26), through the skin(27), as well as by mouth, where the nucleic acid is taken up by cells lining the gut(28). Naked DNA can even be taken up by sperm cells of marine organisms and mammals, and transgenic animals created(29). Geneticists are contemplating using sperms as vectors in gene therapy.
High levels of foreign gene expression was observed in the liver cells of rats, mice and dogs when naked DNA was injected into blood vessels supplying the liver(30). Gene expression is observed in skin cells injected with naked DNA(31), and naked DNA was integrated into chromosomes of cells and expressed in human and pig skin(32). Researchers have found integration of a plasmid-based naked DNA malarial vaccine injected into mouse muscle in a preclinical trial, but dismissed it as "3000 times less than the spontaneous mutation rate for mammalian genome" and hence "not considered to pose a significant safety concern"(33).
One of the key findings is that naked viral DNA is more infectious and have a wider host range than the intact virus. Human T-cell leukaemia viral DNA formed complete viruses when injected into the bloodstream of rabbits(34). Similarly, naked DNA from the human polyomavirus BK (BKV) gave a full-blown infection when injected into rabbits, despite the fact that the intact BKV virus is not infectious(35). This hazard is particularly relevant to the entire range of virus-based gene therapy vectors and naked DNA vaccines under development(36). Modifications to viral genomes can have unexpected effects on virulence and the host range(37)
The safety of gene therapy vectors is unproven(38). The hazards include both direct toxicities and indirect effects (see Box 3) and there is a growing debate over the potential for generating infectious viruses, and harmful effects due to random insertion into the cellular genome(39), both of which are shared by naked DNA vaccines. Recombinant DNA vaccines, in both the naked and intact viral form, also tend to be more unstable and prone to recombination, increasing the likelihood of generating new viruses(40).A viral vaccine made by deleting genes from the simian immuno-deficiency virus (SIV) was found to cause AIDS in infant and adult macaques(41), raising serious safety concerns over similar AIDS viral vaccines for humans.
Hazards from naked nucleic acids
Gene therapy vectors and naked DNA vaccines can cause acute toxic shock reactions (42) and severe delayed immunological reactions(43). Between 1998 and 1999, scientists from US drug companies failed to notify the FDA about six deaths that had occurred during clinical trials of gene therapy, the causes of which are yet to be determined(44). Naked DNA can also trigger autoimmune reactions, in which the bodys immune system attack and kill its own tissues and cells. New research shows that any fragment of double-stranded DNA or RNA introduced into cells can induce these reactions which are responsible for many diseases(45). Examples of autoimmune diseases are rheumatoid arthritis, insulin-dependent diabetes and Graves disease of the thyroid. Many spontaneous mutations are due to insertions of transposable elements and other invasive DNA. Insertion mutagenesis is now found to be associated with a range of cancers, including lung(46), breast(47), colon (48) and liver (49) cancers. Finally, unintended modification of germ-cells may result from gene therapy and vaccinations(50).
Not as much is known concerning naked RNA. It is to be expected that antisense RNA, like antisense DNA, will have biological effects either in blocking the function of homologous genes or genes with homologous domains. RNA may also be reverse transcribed into complementary DNA (cDNA) by reverse transcriptase, which is present in all higher organisms as well as some bacteria(51), to become incorporated into the cells genome.
The direct uptake and incorporation of genetic material from unrelated species is referred to as horizontal gene transfer, or gene transfer by infection, to distinguish it from the usual vertical gene transfer from parent to offspring in reproduction.
Many geneticists may accept that naked nucleic acids are transferred horizontally, especially to microorganisms, but dispute the transfer of transgenic DNA, which they regard to be no different from the host cell DNA.
There is evidence of secondary horizontal transfer of transgenic DNA to soil bacteria and fungi in the laboratory. In the case of fungi, the transfer was obtained simply by co-cultivation(52). Successful transfers of a kanamycin resistance marker gene to the soil bacterium Acinetobacter were obtained using DNA extracted from homogenized plant leaf from a range of transgenic plants: Solanum tuberosum (potato), Nicotiana tabacum (tobacco), Beta vulgaris (sugar beet), Brassica napus (oil-seed rape) and Lycopersicon esculentum (tomato)(53). It is estimated that about 2500 copies of the kanamycin resistance genes (from the same number of plant cells) is sufficient to successfully transform one bacterium, despite the fact that there is six million-fold excess of plant DNA present. A single plant with say, 2.5 trillion cells, would be sufficient to transform one billion bacteria.
Schluter et al(54) investigated horizontal gene transfer under a variety of conditions, some of which gave positive results. For example, a high gene transfer frequency of 5.8 x 10-2 per recipent bacterium was demonstrated for ampicillin resistance transgene - re-isolated from the DNA of transgenic potato - to Erwinia chrysanthem, a bacterial pathogen. This was achieved by 105 copies of the ampicillin resistance gene per potato genome, introduced into 6.4 x 108 bacteria by electroporation. When reduced to one copy of ampicillin resistance gene per potato genome, the gene transfer frequency was still significant at 4 x 10-6. The total genomic DNA from the transgenic potato, estimated to carry two copies of ampicillin resistance gene per potato genome, likewise gave a transfer frequency of 9 x 10-6. With only transgenic potato tissue, it was less than 8.7 x 10-9, effectively nil, according to the limit of sensitivity of the protocol. The same result was obtained by co-cultivation of the transgenic tuber with bacteria for 6 weeks. The negative results were not surprising, given the limited access of the bacteria to plant DNA under those conditions. The authors then calculated an extremely low frequency of gene transfer at 2.0 x 10-17 under extrapolated "natural conditions", assuming the different factors acted independently. The natural conditions are unknown and by the authors own admission, synergistic effects cannot be ruled out.
Free transgenic DNA will be readily available in the rhizosphere around the plant roots, which is an environmental hotspot for gene transfer(55). Gebbard and Smalla(56) have also found evidence of horizontal transfer of kanamycin resistance from transgenic DNA to Acinetobactor, and positive results were obtained using just 100ml of plant-leaf homogenate. Many other factors, such as the density of bacteria, temperature, availability of nutrients, heavy metals and pH, can greatly influence the frequency of horizontal gene transfer in nature(57). Moreover, less than one percent of all bacteria in the environment can be isolated(58) and monitored for horizontal gene transfer, so negative results in the field must be interpreted with due caution. There is no ground to assume that horizontal transfer of transgenic DNA will not take place under natural conditions.
There are also reasons to suspect that transgenic DNA may be more likely to take part in horizontal gene transfer than the organisms own genes (see Box 4)(59).
Reasons to expect that transgenic DNA may be more likely to spread horizontally than non-transgenic DNA
Horizontal gene transfer is uncontrollable. Unlike chemical pollutants which break down and become diluted out, nucleic acids are infectious, they can invade cells and genomes, to multiply, mutate and recombine indefinitely.
Horizontal gene transfer is by no means unknown to our Governments. Among the scientific advice given by the UK Ministry of Agriculture, Fisheries and Food (MAFF) to the US Food and Drug Administration (FDA) at the end of 1998 (62) are the following warnings:
Potential hazards from horizontal gene transfer of naked/free nucleic acids
The dangers of generating new viruses and bacteria that cause diseases, and spreading drug and antibiotic resistance among the pathogens, were both foreseen by the pioneers of genetic engineering. That was why they called for a moratorium in the Asilomar Declaration of 1975. But commercial pressures cut the moratorium short, and guidelines were set up based on assumptions, every one of which has been invalidated by scientific findings since(63). Within the past 20 years, drug and antibiotic resistant infectious diseases have come back with a vengeance. Geneticists have confirmed that the diseases are due to new viral and bacterial strains that have been created by horizontal gene transfer and recombination. Horizontal gene transfer is now recognized to be widespread, involving the entire biosphere, with bacteria and viruses in all environments serving as reservoir and highway for gene multiplication, gene swapping and trafficking. Has genetic engineering contributed to creating the new pathogens, and will it continue to do so through the unregulated release of naked and free nucleic acids? (64) The possible links between genetic engineering biotechnology and the recent resurgence of infectious diseases are summarized in Box 6.
Dormant and relict viral sequences have been discovered in the human and other animal genomes at least 20 years ago(65). Viral sequences have also been discovered recently in plant genomes(66). Viral transgenes are found to recombine with defective viruses to generate infectious recombinants(67). Recombination between exogenous and endogenous viral sequences are associated with animal cancers(68). It is not inconceivable that the cauliflower mosaic viral promoter, which is in practically all first generation of transgenic plants, may recombine with dormant/relict viral sequences in the genome to regenerate infectious viruses(69), in view of the fact that viral promoters have modules in common. Recombination hotspots may be associated with all transcriptional promoters(70), including those of animal viruses, such as the SV40 and cytomegalovirus, used in animal and human genetic engineering(71). This possibility should be addressed by empirical investigations, particularly in view of the recent claim that a significant part of the toxicity of certain transgenic potatoes fed to young rats may be due to the transgenic construct or the transformation process, or both(72).
Possible links between genetic engineering biotechnology and the recent resurgence of infectious diseases
In the light of the existing evidence, the most dangerous naked/free DNA may be coming from the wastes of contained users of GMOs which are discharged into our environment. These include constructs containing cancer genes from laboratories in research and development of cancer and cancer drugs, virulence genes from bacteria and viruses in pathology labs and all kinds of other novel constructs and gene combinations that did not previously exist in nature, and may never have come into being but for genetic engineering.
Despite the growing body of evidence of hazards from the innumerable exotic naked nucleic acids that are created and released in increasing amounts into the environment from the burgeoning biotech industry, there is no effective regulatory oversight, nor is there any indication that our Government is prepared to establish effective regulatory oversight (see Box 7).
Current regulatory oversight is seriously out of date and does not address the dangers of naked or free nucleic acids
The naked/free nucleic acids created by genetic engineering biotechnology are potentially the most dangerous xenobiotics to pollute our environment. Unlike chemical pollutants which dilute out and degrade over time, nucleic acids can be taken up by all cell to multiply, mutate and recombine indefinitely. The need for regulatory oversight at both national and international levels is long overdue. It is irresponsible to continue to exclude naked/free nucleic acids from the scope of the Biosafety Protocol.
Article first published 1999
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