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[SANET-MG] thymine-less death in bacterial gene therapy vectors
June 6, 2006
Prof. Joe Cummins
Thymine-less death in bacterial gene therapy vectors
Thymineless death was first observed fifty four years ago. It is a
peculiar cell death spasm observed when cells are deprived of the DNA
base thymine. The phenomenon is observed in all of the organisms from
bacteria to humans. However, the most detailed information is available
from the study of bacterial auxotrophs ( mutants deprived of essential
chemical building blocks) lacking thymine. When deprived of thymine the
cells accumulate both single and double strand DNA breaks. The double
strand DNA breaks lead to cell death unless promptly repaired (1).
Thymine deprivation is believed to activate a genetic suicide module
leading to DNA degradation and death (2). During thymine starvation the
cells rapidly lose viability but lysates of the cells are capable of
transforming (genetically modifying) recipient cells (3) a kind of sex
Mutant thymine minus bacteria have been prepared as gene therapy vectors
for delivering human genes to patients using a vector that required
thymidine or thymine co-administration with the modified bacteria , once
the vector was excreted into the environment it would suicide thus would
not contaminate the environment with active human genes. In order to
insure that the bacterial vector would not revert to ability to
synthesize thymine the human therapy gene was used to disrupt a gene for
thymine production. Gene disruption is achieved by adding short DNA
tails made up of sequences from the thymine gene to ends of the human
gene. When the human gene with thymine gene tails are injected into the
bacterium the genes tails direct the recombinant gene to the thymine
gene where homologous recombination inserts the human gene into the
thymine gene disrupting the function of the thymine gene and allowing
the human gene to produce its product in the modified bacterium. The
disrupted thymine gene does not revert to its wild form easily. Since
human genes are not readily expressed in the bacterium because of DNA
codon bias , the disrupting gene is inserted as a synthetic
approximation of the human gene. With code adjusted for codon bias(4).
Mouse colitis was treated using Lactococcus lactis modified with a mouse
interleukin 10 (an anti-inflammatory cytokine) gene (5). In that study
the containment of the interleukin gene was not discussed except in a
cursory mention. Pigs were treated with a synthetic interleukin 10 born
in a thymine minus Lactococcus lactis. In that report 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 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 bearing a
thymine gene on a plasmid. Plasmid exchange between recombinant and a
plasmid bearing strain was studied (6) but the plasmids did not appear
to bear a thymine plus gene which would have . complemented the thymine
minus trait to produce a partial diploid that positive for both thymine
production and interleukin 10 production thus capable of growing in an
environment lacking thymine. A phase 1 human trial using Lactococcu
lactis expressing a synthetic human gene for interleukin 10 disrupting a
thymine gene to treat 10 people for Chrohn’s disease was completed in
Holland. Reduction in disease activity was observed and the interleukin
10 producing bacteria recovered in stools were dependent on thymine for
growth (7). The investigators believed that containment of the
transgenic bacteria was complete but there was little effort to recover
partial diploids that were thymine positive and producing interleukin
10. There did not appear to be an effort to recover any bacteria
transformed with the gene for interleukin 10 from the lysates of dead
transgenic therapy bacteria.
The thymine minus trait has also been used to construct a live
attenuated cholera vaccine. A thymine gene was cloned mutated in vitro
then returned to Vibrio cholera to produce the non proliferative strain,
as a vaccine candidate(8). A thymine minus strain of Streptococcus
thermophilus (a bacterium used to produce yogurt and cheese) was
constructed as a vector to deliver transgenes used in food production.
In this case the thymine minus gene selected for use was a spontaneous
Containment of all of the modified thymine minus suicide strains is
dependent upon two important considerations. The first consideration is
appearance of the thymine plus trait in the modified bacteria, that
will, of course suppress the suicide function. Mutational reversion is
unlikely in the disrupted strain but possible in strains bearing a
conventional thymine minus mutation. The thymine plus trait can also be
activated by the introduction of the thymine plus trait on a plasmid or
transducing bacteriophage. Producing a partial diploid. Since the
thymine minus trait is recessive the cell will be thymine positive and
suicide is prevented. This possibility has not been discussed by the
those promoting use of the thymine minus trait for bacterial
containment. There are numerous lacctococcus plasmids , one in
particular thymine plus plasmid is used as a selectable marker in place
of an antibiotic resistance marker (10,11). Cosmids are viruses that
transducer plasmids , the Lactococcus lactis bacteriophage sk1
efficiently transduces plasmids (12). Lactococcus contains numerous
lysogenic bacteriophages many of which are capable of transducing genes.
It appears to be very difficult to eliminate the probability that
thymine plus partial diploids will prevent containment of the thymine
minus strains. The second consideration is related to the fate of the
DNA of dead and dying thymine minus bacteria in the environment. Lysis
of such bacteria will lead to the release of the synthetic interleukin
10 DNA into the bowels or fecal environment 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
(13). Food commensal bacteria have been implicate in the horizontal gene
by natural gene transformation has been implicated in antibiotic
resistance transfer and such transfer may equally spread a synthetic
interleukin 10 gene. Both living, dying and dead bacteria may be sources
of genes for the transfer.
Thymine minus bacteria are being promoted as bacterial vectors for human
gene therapy. In one case stage 1 human trials have been reported.
Unfortunately, the experiments reported, here to fore , seem to have
ignored avenues for the spread of transgenes from the bacteria to the
natural environment. Appropriate control experiments should be done or
the human experiments should be curtailed. It has grown popular to
describe microbial gene therapy as a “probiotic” treatment. Probiotic
treatment has a long and honorable history as an effective and ethical
medical treatment while microbial gene therapy, in contrast, has not yet
been applied enough to measure its benefits or most importantly, its risks.
1. Ahmad SI, Kirk SH and Eisenstark A. Thymine metabolism and
thymineless death in prokaryotes and eukaryotes. Annu Rev Microbiol.
2. Sat B, Reches M and Engelberg-Kulka H. The Escherichia coli mazEF
suicide module mediates thymineless death. J Bacteriol. 2003
3. Bousque JL and Sicard N. Size and transforming activity of
deoxyribonucleic acid in Diplococcus pneumoniae during thymidine
starvation. J Bacteriol. 1976 Nov;128(2):540-8.
4. Stiedler,L,Rottiers,P and Remaut,E. Self containing lactobacillus
strain United States Patent Application 20050276788
5. Steidler L, Hans W, Schotte L, Neirynck S, Obermeier F, Falk W, Fiers
W and Remaut E. Treatment of murine colitis by Lactococcus lactis
secreting interleukin-10. Science. 2000 Aug 25;289(5483):1352-5.
6. Steidler L, Neirynck S, Huyghebaert N, Snoeck V, Vermeire A,
Goddeeris B, Cox E, Remon JP and Remaut E. Biological containment of
genetically modified Lactococcus lactis for intestinal delivery of human
interleukin 10. Nat Biotechnol. 2003 Jul;21(7):785-9.
7. Braat H, Rottiers P, Hommes DW, Huyghebaert N, Remaut E, Remon JP,
van Deventer SJ, Neirynck S, Peppelenbosch MP and Steidler L. A Phase I
Trial With Transgenic Bacteria Expressing Interleukin-10 in Crohn’s
Disease. Clin Gastroenterol Hepatol. 2006 May 19; [Epub ahead of print]
8. Valle E, Ledon T, Cedre B, Campos J, Valmaseda T, Rodriguez B, Garcia
L, Marrero K, Benitez J, Rodriguez S and Fando R. Construction and
characterization of a nonproliferative El Tor cholera vaccine candidate
derived from strain 638. Infect Immun. 2000 Nov;68(11):6411-8.
9. Sasaki Y, Ito Y and Sasaki T. ThyA as a selection marker in
construction of food-grade host-vector and integration systems for
Streptococcus thermophilus. Appl Environ Microbiol. 2004 Mar;70(3):1858-64.
10. Mills S, McAuliffe OE, Coffey A, Fitzgerald GF and Ross RP. Plasmids
of lactococci - genetic accessories or genetic necessities? FEMS
Microbiol Rev. 2006 Mar;30(2):243-73
11. Ross P, O'Gara F and Condon S. Thymidylate synthase gene from
Lactococcus lactis as a genetic marker: an alternative to antibiotic
resistance genes. Appl Environ Microbiol. 1990 Jul;56(7):2164-9.
12. Chandry PS, Moore SC, Davidson BE and Hillier AJ. Transduction of
concatemeric plasmids containing the cos site of Lactococcus lactis
bacteriophage sk1. FEMS Microbiol Lett. 2002 Oct 29;216(1):85-90
13. Lorenz,M and Wackernagel,W. Bacterial gene transfer by natural
genetic transformation in the environment Microbial Reviews 1994,58:563-602
14. Wang HH, Manuzon M, Lehman M, Wan K, Luo H, Wittum TE, Yousef A and
Bakaletz LO. Food commensal microbes as a potentially important avenue
in transmitting antibiotic resistance genes. FEMS Microbiol Lett. 2006
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