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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 after death.

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 mutant (9).

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.

Reference

1. Ahmad SI, Kirk SH and Eisenstark A. Thymine metabolism and thymineless death in prokaryotes and eukaryotes. Annu Rev Microbiol. 1998;52:591-625

2. Sat B, Reches M and Engelberg-Kulka H. The Escherichia coli mazEF suicide module mediates thymineless death. J Bacteriol. 2003 Mar;185(6):1803-7

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 Feb;255(2):328.

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