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[SANET-MG] probiotic gene therapy shouod be banned
ISIS Press Release 03/07/06
GM Probiotic Bacteria in Gene Therapy
Prof. Joe Cummins and Dr. Mae-Wan Ho repeat their call for a ban on GM
probiotics (Ban GM probiotics) as the first clinical trial has been
A fully referenced version of this article is posted on ISIS members’
website. Membership details here
Natural probiotic bacteria promote health, but GM probiotics are
Probiotic bacteria are beneficial bacteria living in the human gut that
are now widely used as food additives for their health-promoting
effects. These bacteria have co-evolved with their human host over
millions of years. Their contributions to health and to the development
of the host’s immune system depend on an intricate web of
bacteria-bacteria and bacteria-host relationships that if thrown out of
balance will most likely result in disease. For that reason alone,
probiotics should never be subjected to genetically modification, let
alone genetic modification for use directly on human beings.
There are other reasons that make GM probiotics particularly hazardous.
The human gut is an ideal environment for horizontal gene transfer and
recombination, the main route to creating dangerous pathogens. And
pathogens created from probiotic bacteria will be pre-adapted to invade
and colonize the human gut. We have published a paper in a scientific
journal expressing our concerns  (GM probiotics should be banned).
Despite our warning, the first clinical trial using GM probiotic
bacteria as gene therapy vector has taken place . And supporters have
written to the journal  criticizing our paper  for
“sensationalism” and “lack of common sense”, and insist on defining
probiotics in such a way as not to exclude GM strains. We took issue
with their assertions  (Reply to GM microbes for human health)
especially in their attempt to blur the distinction between GM and
natural probiotic bacteria, which is misleading and dangerous.
Let’s look at the first clinical trial with GM probiotics and the
research leading up to it and beyond more closely here.
GM probiotic gene therapy uses an old bag of tricks
This first probiotic gene therapy makes use of a bag of tricks –
thymineless death - that was first discovered 50 years ago.
Thymineless death is a peculiar cell death spasm that occurs when cells
are deprived of the DNA base thymine. It happens in all organisms from
bacteria to humans, but the most detailed information is available from
the study of mutant bacteria strains lacking the ability to make
thymine, an essential building block for DNA. When deprived of thymine,
therefore, the cells accumulate both single and double strand DNA
breaks. The double strand DNA breaks lead to cell death unless promptly
repaired . Thymine-deprivation is believed to activate a genetic
suicide module leading to DNA degradation and death . During thymine
starvation, the cells rapidly lose viability. But lysates of the cells
are nevertheless capable of transforming (genetically modifying)
recipient cells , a kind of sex after death.
Mutant thymine-minus bacteria have been prepared as gene therapy vectors
for delivering human genes to patients. In order to make sure that the
bacterial vector would not regain its ability to synthesize thymine, the
human therapy gene was inserted into the bacterial vector so as to
disrupt a gene for thymine synthesis. Gene disruption is achieved by
adding short DNA segments of sequences from the thymine gene to both
ends of the human gene, so as to direct the foreign gene to the thymine
gene where homologous recombination can take place to insert the human
gene into the thymine gene. This allows the human gene to be expressed
in the genetically modified bacterium in place of the thymine gene, and
the disrupted thymine gene does not revert easily.
As human genes are not readily expressed in the bacterium because
different DNA codons for the same amino acids tend to be used (codon
bias), the disrupting gene inserted is a synthetic approximation of the
human gene, with codons adjusted to suit the bacterium .
Mouse model inadequately investigated
Mouse colitis (gut inflammation) was treated using Lactococcus lactis
modified with a mouse interleukin-10 (an anti-inflammatory cytokine)
gene . In that study the containment of the interleukin gene was not
discussed except for a cursory mention. Pigs were treated with a
synthetic interleukin-10 gene in a thymine-minus Lactococcus lactis.
Both mixed bacterial cultures, or bacteria recovered from the pigs ileum
were studied to determine whether or not there was mating to produce a
thymine positive bacteria from the thymine-minus transgenic bacteria
used to treat the pigs. There was no evidence that thymine positive
strains were appearing due to reversion and loss of the interleukin gene.
However, the experiment was not designed to detect ‘partial diploids’
that carry a functioning thymine gene on a plasmid, which could then
enable the bacterium carrying the interleukin gene disupting the thymine
gene on its chromosome to escape cell death. Plasmid exchange between
the transgenic and a plasmid-bearing strain was studied , but the
plasmid did not appear to carry a thymine-plus gene which would have
complemented the thymine-minus trait to produce a partial diploid
positive for both thymine production and interleukin-10 production, and
thus capable of growing in an environment lacking thymine.
Phase 1 clinical trial not adequately followed up
The phase 1 human trial using Lactococcu lactis expressing the synthetic
human gene for interleukin-10 inserted into the thymine gene to treat 10
people for Crohn’s disease was carried out in Holland . A reduction
in disease activity was reported and the interleukin-10 producing
bacteria recovered in stools were found to be dependent on thymine for
But as every microbiologist knows, the proportion of gut bacteria that
can be cultured is very small, certainly not greater than 10 percent,
and little attempt was made to recover partial diploids, or to test
whether the lysate of dead transgenic therapy bacteria could transform
other gut bacteria. Nevertheless, the investigators concluded that
containment of the transgenic bacteria was complete.
The thymine-minus trait is gaining popularity in GM bacteria as a means
of ‘containing’ the transgene. It has also been used to construct a live
attenuated cholera vaccine. A thymine gene mutated in vitro was cloned,
and then returned to Vibrio cholera to produce the non-proliferative
strain as a vaccine candidate . A thymine-minus strain of
Streptococcus thermophilus (a bacterium used to produce yogurt and
cheese) was constructed as a vector to deliver transgenes for food
production; in this case, the thymine-minus gene was a spontaneous
Transgene containment using modified thymine-minus suicide strains is
dependent on two important assumptions, both of which are invalid. The
first is that mutational reversion is unlikely in the disrupted gene
strain, though it is possible in strains carrying a conventional
thymine-minus mutation. In the event of recombination, the transgene has
to be spliced out for reversion to occur, so the transgenic bacterium is
no longer transgenic. However, the strain that has recombined with the
transgenic bacterium and gained the transgene may not be isolatable by
current culture techniques. This could result in a false negative
indicating that the transgene has not escaped, especially if the
interleukin gene is carried on a plasmid in a partial diploid bacterium.
The thymine-plus trait can also be introduced into the transgenic
bacterium itself on a plasmid or a transducing bacteriophage, resulting
in a partial diploid thereby preventing cell suicide. These
possibilities have not been considered or discussed by those promoting
the use of the thymine minus trait for bacterial containment.
There are numerous Lacctococcus plasmids, one in particular, a
thymine-plus plasmid is used as a selectable marker in place of an
antibiotic resistance marker [13,14]. The Lactococcus lactis
bacteriophage sk1 efficiently carries plasmids and transfers them into
cells . Lactococcus contains numerous lysogenic bacteriophages many
of which are also capable of carrying and tranferring genes into cells.
Another assumption is that dead cells will not engage in gene exchange,
or that transgenic DNA from dead cells will not transfer horizontally to
other bacteria. In fact, lysis of transgenic bacteria will release the
synthetic interleukin-10 DNA in the bowels or faeces where the DNA may
transform a range of bacterial species. For example, Lactobacillus may
be transformed at a relatively high frequency in the natural environment
. Food commensal bacteria have been implicated in the horizontal
transfer of antibiotic resistance, and such transfer may equally spread
the synthetic interleukin-10 gene. Living, dying and dead bacteria may
all be sources of gene transfer.
Thymine-minus bacteria are being promoted as bacterial vectors for human
gene therapy. Unfortunately, the experiments reported so far seem to
have ignored the avenues for the spread of transgenes from the bacteria
to the natural environment via well-known processes of horizontal gene
transfer and recombination.
Proponents are now describing microbial gene therapy as “probiotic”
treatment, and actually making use of genetically modified probiotic
bacteria. Probiotic treatment has a long and honourable history of
effective and ethical medical treatment while microbial gene therapy is
an extremely risky business, especially when it uses genetically
modified probiotic bacteria.
We reiterate our call for a ban on GM probiotic bacteria.
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