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 carried out
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 downright
dangerous
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 [1] (GM
probiotics should be banned).
Despite our warning, the first clinical trial using GM probiotic bacteria as
gene therapy vector has taken place [2]. And supporters have written to the
journal [3] criticizing our paper [1] 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 [4] (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 [5]. Thymine-deprivation is believed
to activate a genetic suicide module leading to DNA degradation and death
[6]. During thymine starvation, the cells rapidly lose viability. But lysates
of the cells are nevertheless
capable of transforming (genetically modifying) recipient cells [7],
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 [8].
Mouse model inadequately investigated
Mouse colitis (gut inflammation) was treated using Lactococcus lactis modified with a mouse
interleukin-10 (an anti-inflammatory cytokine) gene [9]. 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 [10], 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 [2]. A reduction in disease activity was reported and the interleukin-10
producing bacteria recovered in stools were found to be dependent on thymine
for growth.
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
[11]. 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 mutant
[12].
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
[15]. 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
[16]. 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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