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Comments on: Environment Assessment: Confined field study of a transgenic pink bollworm, Pectinophora gossypiella

Prof. Joe Cummins, Prof. Emeritus of Genetics, University of Western Ontario. Dr. Mae-Wan Ho, Institute of Science in Sociey

Environment assessment (EA) of the confined field study of a transgenic pink bollworm, Pectinophera gossypiella was prepared by US Department of Agriculture (USDA), Animal and Plant Health Inspection Service (APHIS) to evaluate application for a permit application Jan. 17,2001 from APHIS. EA is designated Docket No. 01-024-1 and the comments below refer to that Docket.

Summary of the comments: The piggyBac transposon used to genetically modify pink bollworm was originally discovered by its ability to infect insect cells and baculovirus and to move between the two. The environment assessment of the pink bollworm release did not discuss the likelihood that baculovirus bearing piggyBac or related transposons could rescue the inactivated piggyBac in the pink bollworm cell by complementation and genetic recombination. The interaction of the piggyBac transposon in the bollworm cell and baculovirus should be studied in the laboratory before the "contained" field trial is allowed. The proposed trial does not "contain" the predictable interaction of baculovirus and pink bollworm. Baculoviruses are known to be efficient vectors for transferring genes into animal and human cells. There is also evidence that piggyBac transposon vectors carrying transgenes are unstable, and undergo secondary mobilization to transfer horizontally, potentially to all species including human beings.

The objective of the proposed research associated with this permit application is to carry out field experiments with pink bollworm (PBW) genetically engineered with a green fluorescent protein (GFP) marker gene derived from a jellyfish. These studies are a prelude to developing genetically engineered sterile PBW for general release. The ability to identify the origin of native moth captures in the San Joaquin Valley of California is paramount to optimizing release strategies for this program. The multiple levels of physical and biological confinement in the proposed field tests are: (1) isolation by distance; (2) isolation by screen cages; (3) reproductive sterilization; (4) removing wings of females and placing them in secondary cages; (5) male pheromone traps; (6) destruction of the cotton that may contain bollworms; (7) flooding the area with a high-ratio of sterilized bollworms; and (8) insecticide treatment, if required.

The transgenic bollworms are genetically modified using the piggyBac transposon. The piggyBac element is a deoxyribonucleic acid (DNA) transposable element capable of integrating into other DNA by a transposase enzyme encoded within the element, only when the Inverted Terminal Repeats (ITR) of the element are intact. In the construct used for transformation of the PBW, the transposase gene of the piggyBac element was destroyed by insertion of an expression cassette containing GFP gene driven by a single copy of the Bombyx mori BmA3 promoter. This manipulation destroys the ability of the construct to move on its own. Transformation was done by co-injecting a helper plasmid along with the donor plasmid carrying the transgene into very young PBW embryos. The donor plasmid contains the transforming construct flanked by piggyBac ITRs. The helper plasmid encodes an intact piggyBac transposase gene but has one of its ITRs missing. Both ITRs are absolutely essential for piggyBac transposase mediated integration. Therefore, the helper plasmid lacking one or the other of the ITS cannot integrate itself into target DNA. The genes used from the donor organism and the piggyBac-derived portions of the vectors used to build the transforming construct were cloned off-site. Specifically, Escherichia coli was the immediate host for the plasmids carrying the cloned genes used to make the transforming constructs. The piggyBac transposable element was first discovered in cabbage looper cell culture at the University of Notre Dame (Fraser et al., 1995; 1996; Wang and Fraser 1993).

The GFP is used as a marker to rapidly identify male moths used in the sterile moth control program. Later, the piggyBac vector may be used to insert female killing genes carried by GM male moths to eradicate the bollworm pest. The green fluorescent marker may pose no major threat to the environment should the gene escape, but its incorporation into the cells of non-target organisms including human should be studied.

PiggyBac was discovered by its genetic impact on the baculovirus chromosome, and by its ability to move from insect to baculovirus (Fraser et al 1995; 1996, Wang and Fraser 1993). But no laboratory experiments were submitted by APHIS to deal with the potential rescue of the inactivated piggyBac transposon by active ones carried in baculovirus. APHIS considered the potential rescue of inactivated piggyBac by related transposons in the bollworm genome, and that possibility was dismissed. APHIS failed to consider the interaction of inactivated piggyBac with baculovirus in the environment of the field test. The field "containment" of the GM bollworm was totally uncontained regarding exposure of the GM bollworms to soil borne baculovirus bearing active piggyBac transposons or transposons capable of rescuing the inactive GM piggyBac carried by the bollworm. APHIS should have required extensive laboratory experiments using a range of baculovirus, including strains with homologous GM piggyBac to provide good estimates of GM piggyBac escape through recombination mediated by baculovirus.

Rescue of the inactivated GM piggyBac transposon in the pink bollworm by piggyBac or related transposons in baculovirus can be achieved by complementation, homologous recombination, gene conversion or illegitimate recombination. APHIS seems to have ignored the obvious interaction between the soil borne virus and the test bollworm and made no effort to contain or monitor the virus interaction. The virus, normally soil borne, can easily enter and leave the "containment" area proposed by APHIS as wind borne dust or as clods on the shoes or gloves of workers. Certainly, APHIS bears the burden of proving the experiments to be safe and to protect the environment.

Adequate laboratory studies must be done prior to the field release of potentially dangerous GM arthropods. Such experiments must include serious efforts to rescue the inactivated GM piggyBac using a range of baculovirus strains bearing piggyBac and baculovirus bearing transposons such as tagalong (Bauser et al 1999).

Ecological considerations for the impact of recombinant baculovirus insecticides have been studied extensively (Richards et al 1998). The study emphasized baculovirus containing scorpion toxin. Impact on non-target insects is extrapolated from insects of related phylogeny, a practice difficult to defend. The recombinant baculovirus was very persistent, and can reshape an entire ecosystem. Modification of baculovirus host range specificity has been achieved by inserting or deleting genes (Theim 1997).

USDA has two patents related to baculovirus. Patent US6162430: Insect control with multiple toxins, and US5639454: Recombinant baculovirus with broad host range. These patents may be relevant to the application on GM pink bollworm but do not explain why APHIS did not discuss baculovirus in the EA.

Baculovirus vectors efficiently transfer genes into human liver cells (Hofmann et al 1995; Boyce and Bucher 1996). The vectors transferred into human liver tissues most effectively in perfused liver tissue because serum components hampered virus transfer (Sandig et al 1996). Human conditions associated with defects in complement should allow liver transfer of recombinant baculovirus. Inhibitors of complement facilitate baculovirus gene transfer (Hofmann and Strauss 1998). Hybrid baculovirus-adenovirus vectors have been used to deliver genes to human cells (Palombo et al 1998). Baculovirus vectors have been used to deliver hepatitis B to human liver efficiently to allow study of hepatitis B drug therapy (Delaney et al 1999). Recombinant piggyBac rescued by baculovirus could infect humans with untoward consequences.

Another problem is that the integrated, disabled piggyBac vector is already known to be unstable in the silkworm, probably because it can be mobilized by the transposase of transposons already in the host genome (Toshiki et al, 2000, Ho and Cummins, 2001a, Appendix 1). That means the horizontal spread of piggyBac-borne genes are inevitable, with the potential for insertion mutagenesis and insertion carcinogenesis for animals and human beings. If female-killing genes are eventually incorporated into the piggyBac constructs, the impacts on biodiversity could be devastating (see Ho and Cummins, 2001b, Appendix 2).

References

  1. Bauser,C,Elick,T and Fraser,M "Proteins from nuclear extracts of two lepidopteran cell lines recongnize the ends of TTAA-specific transposons piggyBac and tagalong" 1999Insect Mol Biol 8,223-30
  2. .Boyce,F and Bucher,N "Baculovirus-mediated gene transfer into mammalian cells" 1996Proc. Natnl Acad Sci USA 93,2348-52
  3. Delaney,W,Miller,T, and Isom,H "Use of the hepatitis B virus recombinant baculovirus-Hep G2 system to study the effects of beta 2',3' dideoxy 3'thiaceydine on replication of hepatitis B virus and accumulation of covalently closed circular DNA"1999 Antimicrob Agents Chemother 43,2017-26
  4. Fraser, M.., Cary,L Boonvisudhi,K and. Wang.G " Assay for movement of lepidopteran transposon IFP2 in insect cells using a baculovirus genome as a target DNA."1995 Virology 211: 397-407.
  5. Fraser, M. Ciszczon, T. Elick,T and Bauser C " Precise excision of TTAA specific lepidopteran transposons piggyBac (IFP2) and tagalong (TFP3) from the baculovirus genome in cell lines from two species of Lepidoptera"1996 Insect Mol Biol 5,141-151.
  6. Ho MW and Cummins J. "Stop the release of GM insects" I-SIS News 9 June 2001a, ISSN: 1474-1547 (print) ISSN: 1474-1814 (online) www.i-sis.org.uk
  7. Ho MW and Cummins J. "Terminator insects - the killing of females" I-SIS News 9 June 2001b, ISSN: 1474-1547 (print) ISSN: 1474-1814 (online) www.i-sis.org.uk
  8. Hofmann,C and Strauss,M "Baculovirus mediated gene therapy in the presence of human serum or blood facilitated by inhibition of the complement system" 1998 Gene Ther 5,531-6
  9. Palombro,F,Mociotti,A,Recchia,A,Cortese,R,Ciliberto,G and LaMonica,N "Site specific integration in mammalian cells mediated by a new hybrid baculovirus-adeno-associated virus vector" 1998 J Virol 72,5025-34
  10. Richards,A,Matthews,M and Christain,P "Ecological considerations for the environmental impact evaluation of recombinant baculovirus insecticides" 1998Ann Rev. Entomol 43,493-517
  11. Sandig,V,Hofmann,C,Steinert,S,Jennings,Gschlagg,P and Strauss,M "Gene transfer into hepatocytes and human liver tissue by baculovirus vectors" 1996 Human Gene Ther 20,1937-45
  12. Thiem,S "Prospects for altering host range for baculovirus bioinsecticides" 1997 Curr Opin Biotechnol 8,317-22
  13. Toshiki T, Chantal T, Corinne R, Toshio K, et al. Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nature Biotechnology 2000: 18: 81-84.
  14. Wang, H., and J. Fraser. " TTAA serves as the target site for TFP3 lepidopteran transposon insertions in both nuclear polyhedrosis virus and Trichoplusia ni geneomes."1993 Insect Mol Biol (1): 109-16.

Appendix 1

(Reprint from ISIS News 9/10, June 2001, ISSN: 1474-1547 (print) ISSN: 1474-1814 (online) )

Stop the Release of GM Insects!

The United States Department of Agriculture (USDA) received an application to release GM pink bollworm engineered with jumping genes this summer. This is now delayed as a result of legal challenge from civil society. Dr. Mae-Wan Ho and Prof. Joe Cummins expose evidence of instability in these GM insects, and warn of rampant horizontal gene transfer and recombination. Releasing such GM insects is tantamount to giving wings to the most aggressive genome invaders. They argue that the project should not be allowed to go ahead.

The release was planned to begin July 15 2001 and end July 14 2002 in a small field in Arizona. The pink bollworms are engineered with a jumping gene carrying a green flourescent protein (GFP) from a jelly-fish. The purpose of the release experiment is to use the GFP marker to evaluate the efficacy of sterile insects that are being developed in the hope of eradicating the bollworm pest. The release is being challenged by the International Center for TechnologyAssessment and Center for Food Safety in DC (www.centerforfoodsafety.org), The application can be viewed at: http://www.aphis.usda.gov/biotech/arthropod/permits/0102901r/0102901r.html, and the public are invited to comment by July 23. So please do so! Our detailed submission is posted on I-SIS Website, "Comments on: Environment Assessment: Confined field study of a transgenic pink bollworm, Pectinophora gossypiella" <www.i-sis.org.uk>).

Geneticists have created a particularly hazardous class of gene transfer vectors for engineering insects. These are transposons, or mobile genetic elements, which, as the name implies, are genetic units that can move from one site to another in the same genome or move between genomes belonging to unrelated species. Transposons are related to viruses and proviral sequences that, like transposons, are found in the genomes of all species.

A transposon consists of several genes flanked by terminal repeat sequences. One of the genes will code for the enzyme transposase, which is necessary for moving the element. However, elements that have lost the transposase gene can nevertheless get help from the enzyme encoded in other transposons. Transposons come in groups, or superfamilies, many of which have members distributed widely across species belonging to different phyla of both animals and plants. These 'promiscuous' transposons have found special favour with genetic engineers, whose goal is to create 'universal' systems for transferring genes into any and every species on earth. Almost none of the geneticists has considered the hazards involved.

A group in Boston created a vector from mariner, a superfamily of transposons found across genomes of diverse species from insects to plants and vertebrates, including human beings. One element belonging to this superfamily, Hirmar1, isolated from the horn fly, was used to make 'minitransposons' consisting of the short inverted terminal repeats, between which any gene expression cassette(s) can be inserted. The researchers constructed a minitransposon with a kanamycin antibiotic resistance marker gene driven by a bacterial promoter. This minitransposon was found to jump easily into the E. coli and Mycobacterium chromosome. It is known to recognize the dinucleotide TA. The probability of this dinucleotide occurring in any stretch of DNA is 0.252 or 6.25%. Within the 500 base pairs of the bacterial chromosome analysed, 21 of the 23 possible TA dinucleotide insertion sites were occupied [1].

The experiment shows that the transposon can be stripped down to the bare minimum of the flanking repeats, and it can still jump into genomes. The reason, as mentioned earlier, is that the transposase function can be supplied by a 'helper' transposon. Such helper transposons are ubiquitous. So, it would seem obvious that integrated transposon vectors may easily jump out again, to another site in the same genome, or to the genome of unrelated species. There are already signs of that in the transposon, piggyBac, used in the GM bollworms to be released by the USDA this summer.

The piggyBac transposon was discovered in cell cultures of the moth Trichoplusia, the cabbage looper, where it caused high rates of mutations in the baculovirus infecting the cells by jumping into its genes [2]. The piggyBac is 2.5kb long with 13 bp inverted terminal repeats. It has specificity for sites with the base sequence TTAA. (The probability of this sequence occurring is 0.254 or 0.4%.) This transposon was later found to be active in a wide range of species, including the fruitfly Drosophila, the mosquito transmitting yellow fever, Aedes aegypti, the medfly, Ceratitis capitata, and the original host, the cabbage looper [3]. The piggyBac vector gave high frequencies of transpositions, 37 times higher than mariner and nearly four times higher than Hirmar.

In another experiment, the piggyBac vector, with its transposase gene disabled and carrying the green fluorescent protein gene cassette, was used to transform the silkworm, Bombyx mori L [4]. Transposase function was provided by a helper-plasmid containing a piggyBac transposon also disabled, by having one of its terminal repeats removed. The integrative vector and helper plasmids were both injected into silkworm embryos. The adult fertile moths (G0) resulting from the injected embryos were mated in single pairs among themselves or backcrossed to the unmodified parent, and the resultant broods (G1) were analysed.

A total of 2498 embryos from two strains of silk worms were injected, 1164 (46.6%) of the embryos hatched resulting in 654 (26.7%) fertile adults, single-pair matings among which 12 broods (0.5%) expressing green fluorescent protein were found.

The genomic DNA of the broods were analysed with Southern blot (a technique that gives information on the inserts). Here is how the authors reported their results.

"Southern blot analyses of the DNA of transformed G1 insects showed that one to three different inserts were present in a single animal and that larvae from the same progeny [ie, brood] had different insertions. These insertions were inherited independently at the G2 generation…

"The presence of multiple independent inserts in many G1 larvae indicates that a single gamete from the G0 parent can harbor several insertions and that different gametes can have different insertions. Eighteen insertions were observed in 12 G1 individuals issued from three transformed parents. It is likely that this result underestimates the total number of insertion events that occurred in the G0 moths." (p.82)

Why were there such a large number of different inserts? There were two possible explanations.

"Either the integration events [of the piggyBac vector] in the germ line occurred late during development [of the injected embryo]", so that the same adult carries a population of germ cells each with different insertions, "or successive rounds of transposition took place after an initial insertion event". The latter hypothesis, considered more likely, "would explain why - despite the low frequency of insertion in the parental population [0.5%] - the number of inserts is high in the transformed insects….. A similar situation was also observed in transgenic C. capitata, and it was also attributed to secondary mobilizations of an initial single insert." (p.82)

In other words, there is evidence that the inserts had moved between the G0 and the G1 generations, and possibly, again between the G1 and G2 generations. The "stable germ line transformation" claimed (p.83) is based on a dangerous instability of the insert, which is prone to secondary mobilisation.

The proposal for the field test is based on the belief that the piggyBac gene inserts would be stable. But there is already evidence, described above, that they are not stable. Furthermore, piggyBac may be carried by the baculovirus that infect insects, and such virus certainly produces transposase that can move the gene-bearing transposon. The virus can also act as a vector for rapid spread of the modified transposon to a variety of insects.

The proposal claims that no human health concerns are involved in the field trail. It argues "Lepidoptera, in general, do not pose a threat to human health and welfare and should remain a guiding principle in deciding on human risks related to their genetic manipulation." However, the same piggyBac transposon was also used for gene transfer in the mosquito that transmits yellow fever. Worse still, baculovirus, which harbours piggyBac transposon, is used in human gene therapy because it is so good at getting into human cells, so any piggyBac it carries will be efficiently smuggled along.

These artificial transposons are already aggressive and promiscuous genome invaders, and putting them into insects is to give them wings, as well as sharp mouthparts for efficient delivery to all plants and animals and their viruses. The predictable result is rampant horizontal gene transfer and recombination across species barriers. The unpredictable unknown is what kinds of new deadly viruses might be generated [5], and how many new cases of insertion mutagenesis and carcinogenesis they may bring [6]. It is the height of folly and irresponsibility to release such GM insects, let alone GM insects carrying female-killing genes (see "Terminator insects - the killing of females", this issue).

There is a compelling case for stopping these developments altogether on the basis of hazards that can already be foreseen.

  1. Rubin EJ, Akerley BJ, Novik VN, Lampe DJ, Husson RN, and Mekalanos JJ. In vivo transpostion of mariner-based elements in enteric bacteria and mycobacteria. Proc. Natl. Acad.Sci USA 1999: 96: 164-1650. See also "Can such rampant gene shuffling be safe?" Mae-Wan Ho and Angela Ryan, I-SIS News 4, March 2000
  2. See "Terminator insects - a primer" by Joe Cummins, I-SIS Report, March 2001
  3. Lobo N, Li X and Fraser Jr. MJ. Transposition of the piggyBac element in embryos of Drosophila melanogaster, Aedes aegypti and Trichoplusia ni. Mol Gen Genet 1999: 261: 803-10.
  4. Toshiki T, Chantal T, Corinne R, Toshio K, et al. Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nature Biotechnology 2000: 18: 81-84.
  5. See "Genetic engineering superviruses" by Mae-Wan Ho, I-SIS Report, March 2001
  6. See "Unregulated hazards, 'naked' and 'free' nucleic acids" I-SIS Report, Jan. 2000

Appendix 2

(Reprint from ISIS News 9/10, June 2001, ISSN: 1474-1547 (print) ISSN: 1474-1814 (online) )

Terminator Insects - The Killing of Females

GM pink bollworms are a prelude to developing female-killing traits to control bollworm pests. Dr. Mae-Wan Ho and Prof. Joe Cummins explain the genetics and hazards of female killing systems.

The female-killing systems developed out of the sterile insect technique (SIT), which has been used in biological pest control since the 1950s. It involves mass rearing and release of insects made sterile with X-irradiation and other methods. Initially, sterile insects of both sexes were released, but sterile females were thought to be detrimental to pest control. One main reason is that a single male can mate with a large number of females, while each female will mate with only a few males. Hence, genetic sorting mechanisms (GSMs) were invented to kill the females. Until quite recently, all GSMs have involved radiation-induced X-Y translocation, ie moving of part of a normal X chromosome to the Y chromosome. The resultant Y chromosome then acts as a dominant selectable marker in a population in which all the X chromosomes carry a gene that is 'conditional lethal' in double dose. A conditional lethal is a gene that kills only under certain 'non-permissive' conditions, as for example, exposure to heat. Thus, when the population is heat shocked, the males (XY) survive, while the females (XX) die.

Recently, other conditional lethal systems have been considered for kill off females. Such systems could be introduced into any insect pest species with the help of genetic engineering. As a result, the GM insects could be released directly without pre-sterilisation. One method involves creating a strain that carries a conditional, sex-specific lethal gene, ie, a lethal gene that is expressed only in one sex under 'non-permissive' conditions. The design is such that the non-permissive condition is one that is normally found in nature, whereas the permissive condition (one that permits survival) depends on certain chemicals that could be added to the diet in the insect factory. Researchers have constructed such a system in Drosophila.

They make use of special transcription control elements (ie promoters) and transcription factors, proteins that bind to promoters to enhance transcription [1]. First, the transcription factor, tTa, a protein that interacts with tetracycline, is placed under the control of a promoter, Yp3, which is active in female larvae and adults, but not in males. Next, a reporter gene lacZ, coding for b-galactosidase is placed under another promoter, the tetracycline responsive element, tRe. In the absence of tetracycline, tTa binds to tRe causing the reporter gene to become expressed. In the presence of tetracycline, however, the tetracycline binds to tTa, thereby preventing it from binding to tRe, and the reporter gene is not expressed. (Note: the convention is that genes are in italics, whereas the corresponding gene products are non-italics.)

Strains of flies homozygous for the constructs, Yp3-tTa and tRe-lacZ, respectively were crossed with each other. The resulting progeny were raised in the presence and absence of tetracycline in the culture medium. Adults were stained for b-galactosidase activity. Females grown on normal diet without tetracycline stained strongly for the enzyme, whereas females raised on tetracycline and all males were negative.

To engineer the killing of females, a toxic gene product, Ras64B, was placed under the control of tRe; and a line with tRe-Ras64B was constructed. (Ras1 is a gene that codes for a protein that plays a key role in regulating transcription in the cell. Ras 64B is a defective mutant allele of Ras1. Ras homologues are oncogenes contributing to human cancer.) The tRe-Ras64B line was crossed with another line in which the tTa was placed under a nonspecific (constitutive) promoter. The progeny grown on tetracycline were viable and fertile. On normal medium, however, no progeny survived, ie, both males and females died. When the tRe-Ras64B line was crossed to the Yp3-tTa line, the male progeny but not the female survived in the absence of tetracycline.

Subsequently, the researchers constructed another line homozygous for both Yp3-tTa and tRe-Ras64B on the same chromosome, which was maintained on medium with tetracycline to inhibit the expression of Ras64B. When the flies were transferred to medium without tetracycline, no female progeny were recovered in a sample of more than 5000 males. The genetic system also worked with gene products that are specifically toxic to females.

The males were fertile when mated to other females. This is important for spreading the female-killing gene throughout the pest population. However, it would also spread the gene to related species. The potential also exists for horizontal transfer to unrelated species.

The proposal to engineer these genes into promiscuous transposon vectors will greatly multiply the risks of horizontal transfer to unrelated species, with potentially disastrous effects on biodiversity through the killing of females.

Thomas DD, Donnelly CA, Wood RJ and Alphey LS. Insect population control using a dominant, repressible, lethal genetic system. Science 2000: 287: 2474-6.

Signed:

Prof. Joe Cummins
Dr. Mae-Wan Ho

Article first published 04/07/01



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