Unregulated Hazards Naked and Free
ISIS Report -Produced for the Third World Network
Mae-Wan Ho, Angela Ryan Biology Department, Open
University, Walton Hall, Milton Keynes MK7 6AA, UK
J.Cummins Department of Plant Sciences, University of Western Ontario
T. Traavik Dept. of Virology, Institute of Medical Biology,MH-Breivika And
Norwegian Institute of Gene Ecology, N-9037 Tromso, 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 and free nucleic acids
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
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
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)
yeast (YAC) plasmid (PAC) and mammalian (MAC) made from telomeric and
centromeric repeat sequences(7)
transgene cassettes, often include antibiotic resistance gene
PCR amplified sequences
hairpin-forming oligodeoxynucleotides used in gene therapy(8)
RNA - based
Antisence RNA used in
Ribozymes used in gene
(linked to RNA-dependent RNA polymerase) used in gene therapy(10)
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
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:
Transgenic wastes from
genetically engineered microorganisms in contained use
Transgenic wastes from cell
cultures in contained use
Transgenic wastes from
genetically engineered crops
Transgenic wastes from
genetically engineered fish and other aquatic organisms
Transgenic wastes from
genetically engineered farm animals
Unprocessed transgenic food
and animal feed
Processed transgenic food
for human use and animal feed
textiles such as cotton
Transgenic dust from food
DNA persists in all environments
and is readily taken up by cells of all organisms
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
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).
Hazards of naked nucleic acids
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
Acute toxic shock from
Immunological reaction from
Autoimmune reactions from
double-stranded DNA and RNA
with gene function from anti-sense DNA, RNA and ribozymes
Generation of virulent
Genetic contamination of
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
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.
The horizontal transfer of
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
The mechanisms enabling
foreign genes to insert into the genome also enable them to jump out
again, to re-insert at another site, or to another genome.
The integration sites of
most commonly used artificial vectors for transferring genes are recombination
hotspots, prone to break and join up with other DNA, and so
have an increased propensity to transfer horizontally.
Viral promoters, such as
that from the cauliflower mosaic virus, widely used to boost the
expression of transgenes, also contain a recombination hotspots(60),
and will therefore further enhance horizontal gene transfer.
The unnatural gene
combinations in transgenic DNA tend to be unstable, and hence prone
to recombine and transfer horizontally.
The metabolic stress on the
host organism due to the continuous over-
expression of transgenes
may contribute to the instability of the insert, as it is well-known
that mobile genetic elements in all genomes are mobilized to jump
out of genomes during conditions of stress, to multiply and/or
reinsert randomly at other sites resulting in many
insertion-mutations. The foreign gene-constructs and the vectors
into which they are spliced, are typically mosaics of DNA sequences
from numerous species and their genetic parasites; that means they
will be more prone to recombine with, and successfully transfer to,
the genomes of many species and their genetic parasites(61).
The hazards of horizontal gene
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
Transgenic DNA can spread to
farm workers and food processors via dust and pollen.
Antibiotic resistance marker
genes may spread to bacteria in the mouth, as the mouth contains
bacteria that readily take up and incorporate foreign DNA (see above).
Similar transformable bacteria are present in the respiratory tracts.
Antibiotic resistance marker
genes may spread to bacteria in the environment, which then serves as a
reservoir for antibiotic resistance genes.
DNA is not readily degraded
during food processing nor in the silage, hence transgenic DNA can
spread to animals in animal feed.
Foreign DNA can be delivered
into mammalian cells by bacteria that can enter into the cells.
The ampicillin resistance gene
in the transgenic maize undergoing farm-scale field-trials
in the UK and elsewhere is very mutable, and may compromise treatment
for meningitis and other bacterial infections, should the gene be
transferred horizontally to the bacteria. The potential hazards of
horizontal gene transfer are unlike those we have ever experienced (see
Potential hazards from
horizontal gene transfer of naked/free nucleic acids
Generation of new
viruses that cause disease
Generation of new
bacteria that cause diseases
Spreading drug and
antibiotic resistance genes among the viral and bacterial
pathogens, making infections
Random insertion into
genomes of cells resulting in harmful effects including cancer
Reactivation of dormant
viruses, present in all cells and genomes, which may cause diseases
ecological impacts due to all the above
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
Horizontal gene transfer is
responsible for creating new viral and bacterial pathogens and for
spreading drug and antibiotic resistance
Experimental evidence of
horizontal gene transfers, some between very distant species, has
been obtained in all natural environments and in the gut. Thesewere
all accomplished with artificially constructed vectors used in
Genetic engineering makes
extensive use of antibiotic resistance genes as selectable markers,
thereby increasing the spread of antibiotic resistance genes.
Antibiotics increases the
frequency of horizontal gene transfer 10 to 10000 fold, thereby
enhancing the spread of disease-causing genes as well as antibiotic
strains of bacteria, supposed to be biological contained,
are nevertheless found to survive in the environment, and to swap
genes with other bacteria.
DNA released from dead as
well as live cells are not entirely broken down in the environment,
nor in the gut, where it may be taken up and incorporated into the
genomes of bacteria.
DNA from viruses is more
infectious than the intact virus itself.
inactivation of genetically engineered microorganisms prior to
disposal in the general environment may be ineffective, leaving a
substantial proportion of viruses and bacteria in an infective
Current legal limits of tolerated
releases of genetically engineered microorganisms from
contained use vastly exceed the minimal infective dose of pathogens
and potential pathogens.
transformed into pathogens by horizontal gene transfer of unit
blocks of virulence genes.
Horizontal gene transfers
are bi-directional. Released non-pathogens can be readily converted
into pathogens in one step, by acquiring unit-blocks of virulence
Genetic engineering is
based on facilitating horizontal gene transfer between distant
species by constructing artificial vectors that break down species
The artificial vectors
constructed for genetic engineering are combinations of viral
pathogens and other invasive genetic elements that can generate new
cross-species viral pathogens.
The artificial vectors and
other constructs for genetic engineering are inherently unstable and
prone to recombination, thereby enhancing horizontal gene transfer
vectors made by genetic engineering are essentially
unstoppable, as they contain signals for transfer and replication in
different species; and helper functions for mobilization and
transfer can be supplied by viruses and other genetic parasites
which occur naturally in bacteria in all environments.
The accelerated emergence
of infectious diseases and of drug and antibiotic resistance
coincide with the development of commercial genetic engineering
Many horizontal gene
transfer events responsible for the spread of virulenc and
antibiotic resistance are recent, as inferred from the high degree (>99%)
of similarity in sequences of genes found in unrelated species.
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 beingbut 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).
oversight is seriously out of date and does not address the dangers of
naked or free nucleic acids
there is no monitoring for
horizontal gene transfer in current field trials, including the
farm-scale field trials supported by the Government
there is no requirement for
industry to monitor for horizontal gene transfer in seeking approval
for field trials or commercial release
there is no requirement for
industry to monitor for horizontal gene transfer prior or subsequent
to discharge of transgenic wastes from contained use into the
current regulation allows
certain live genetically engineered bacteria to be discharged into
the environment without any pretreatment
undegraded transgenic DNA
from killed bacteria and viruses are routinely discharged into the
there is no requirement for
industry to report on health impacts of transgenic DNA, which may
spread drug and antibiotic resistance among pathogens, create new
viral and bacterial pathogens as well as cause cancer; nor is there
any monitoring of such health impacts being conducted by our
Nucleic acid sequences and
constructs, including artificial viral vectors, and other genetic
parasites are not subject to regulation, and may be freely
discharged into the environment
current regulation of
contained use still regard DNA as an ordinary, non-hazardous
chemical which can be released unchecked into the environment
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.
Traavik, T. (1999a) Too early may be too late: Ecological risks
associated with the use of naked DNA as a biological tool for research,
production and therapy. pp 29-31. Reported to the Directorate of Nature
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