Can GM Mosquitoes Eradicate Dengue Fever?

A risky strategy experimented on humans without informed consent, and based on data from unpublished studies. Prof. Joe Cummins

What is dengue?

Dengue fever is an illness caused by an RNA flavivirus spread by the bites of Aedes aegypti mosquitoes. The symptoms include fever, headache, rash, severe pains in the muscles and joints, and pain behind the eyes. Dengue fever is rarely fatal, while the related dengue haemorrhagic   fever is a severe disease that leads to death in approximately 5 percent of cases. Dengue hemorrhagic fever is seen most often in children younger than 15 years old. It is also seen most often in individuals with fever and gastrointestinal symptoms followed by capillary haemorrhage [1] (Terminator Mosquitoes to Control Dengue? SiS 39). Incidence of dengue has grown dramatically around the world in recent decades. Some 2.5 billion people – two fifths of the world's population – are now at risk from dengue. The World Health Organization estimates there may be 50 million dengue infections worldwide every year [2]. For more details, see Box.

Terminator mosquitoes tested on mosquitoes and people

Genetically modified (GM) OX513A  Aedes aegypti male mosquitoes are being released in an area inhabited by people. The living modified organism (LMO) released contains a fluorescent molecular marker and a ‘self-limiting’ genetic construct. When female Aedes aegypti mosquitoes mate with OX513A male mosquitoes, all offspring will die, hence preventing the emergence of the next generation. OX513A male Aedes aegypti mosquitoes, developed by Oxitec Ltd. in Oxford, UK, carry a late-acting dominant-lethal gene [1]. The dominant-lethal gene requires only one copy to work, and is expressed conditionally. In the case of the Oxitec creation, the condition that acts to kill the mosquito larvae bearing a dominant gene is absence of the antibiotic tetracycline. The males reared for terminating their larvae offspring when mated with wild type females are maintained on antibiotic-containing media.  The males are homozygous diploids for OX513A so that all of their offspring are doomed in the absence of antibiotic. The lethal factors are active in late stages of larval development [3].

The field test is being done by Oxitec Limited, Oxford, United Kingdom in Grand Cayman. The Cayman Islands are a parliamentary democracy with judicial, executive and legislative branches. The present constitution, which came into effect on 6 November 2009, makes the Cayman Islands a British Overseas Territory.  Results of the Oxitech experiments claiming to prove the safety and benefits of the terminator mosquito have been reported at a press conference in London, UK [4], and at a meeting of the American Association of Tropical Hygiene in Atlanta, Georgia, USA [5], but have not yet appeared in a peer-reviewed journal. As the Cayman Island is under British jurisdiction, the human experimentation involving exposure to GM mosquito appeared to have been undertaken without informed consent as would be required by British law.

Construction of the GM mosquitoes

The OX513A mosquito was constructed using a transposon (jumping gene) piggyBac LA513 isolated from Baculovirus, a common soil dwelling DNA plant virus. LA513 is a non-autonomous piggyBac-based transposon of 8.4 kb. Transgenic mosquitoes are readily identified by red fluorescence due to expression of DsRed2, a red fluorescent protein from a marine microbe. The GM construct contains tTAV, coding for a tetracycline-repressible transcriptional activator, under the control of its binding site, tetO, a minimal promoter from Drosophila heat shock protein 70, and a 3' UTR (untranslated repeat) sequence from another Drosophila gene fs(1)K10. In the absence of tetracycline, tTAV binds to tetO and drives the expression of more tTAV, in a positive feedback loop. In the presence of tetracycline, tTAV binds tetracycline; the tetracycline-bound form does not bind tetO and so no more tTAV is expressed. Consequently, this construct gives very high levels of expression of tTAV in the absence of tetracycline, but only low, basal expression in the presence of tetracycline. High level expression of tTAV is toxic, possibly due to interactions of its VP16 domain in transcription activation of lethal genes, so this construct provides a tetracycline-repressible lethal system [3]. The VP16 domain is the transcriptional activation domain of the herpes virus protein VP16, and is a common feature of transcription activation proteins.  It seems clear that the absence of the antibiotic triggers enhanced transcription factors leading to lethality; however, the final product(s) causing late death at the larvae-pupae boundary have not been identified, which surely should have been done before people are exposed to the transgenic mosquitoes.

The piggyBac transposon and horizontal gene transfer

The piggyBac transposon was discovered in cell cultures of the moth Trichopulsia, also known as the cabbage looper. The looper cells caused extensive mutation in an insect Baculovirus. The transposon is being used extensively as it acts as a gene transfer vector for a number of unrelated insects. Transposons are related to viruses but lack the ability to be packaged in a virion. The piggyBac transposon is flanked by short inverted repeats that initiate non-replicative insertion into the insect chromosome, mediated by an enzyme called transposase that targets the sequence TTAA on the chromosome. When the transposon inserts into a gene sequence, it may cause a mutation, or the transposon may act as a recombination site leading to duplication and deletion of sequences in the chromosomes.

The transposon is usually multiplied in the laboratory by being inserted in a bacterial plasmid. The transposon-bearing plasmids are injected into early insect embryos, where they may insert into the genome of germ cells and become maintained in a lineage arising from the germ cells. The plasmids injected into an embryo include the transposon carrying the gene(s) to be transferred and lacking a transposase, to ‘disable’ the transposon and prevent it from moving by itself. The transposase function is provided by a helper plasmid that has a transposon with the transposase gene, but is otherwise also disabled because it has one terminal short repeat missing, so it cannot insert into the chromosome [6] (piggyBac a name to remember, I-SIS report).

The major hazard of horizontal transfer of the piggyBac transposon has been thoroughly addressed in a previous submission to the USDA objecting to the release of the pink bollworm in 2001 and again in our recent submission objecting to the release of the current transgenic .mosquito [1]. We provided evidence that the disabled vector carrying the transgene, even when stripped down to the bare minimum of the border repeats, was nevertheless able to replicate and spread, basically because the transposase function enabling the piggyBac inserts to move can be by transposons potentially present in all genomes, including that of the mosquito. The main reason for using transposons as vectors in insect control is precisely because they can spread the transgenes rapidly by ‘non-Mendelian' mean within a population, i.e., by replicating copies and jumping into genomes, including those of the mammalian hosts. Although each transposon has its own specific transposase enzyme that recognizes its terminal repeats, the enzyme can also interact with the terminal repeats of other transposons, and evidence suggest “extensive cross-talk among related but distinct transposon families” within a single eukaryotic genome.

The use of the piggyBac transposon has been plagued by problems of instability in transformed Aedes aegypti [7]; and large unstable tandem inserts of the piggyBac transposon were prevalent [8]. In spite of instability and resulting genotoxicity, the piggyBac transposon has been used extensively also in human gene therapy [9]. A number of human cell lines have been transformed, and even primary human T cells, using piggyBac [10]. The piggyBac transposon was found to induce genome wide insertion mutations disrupting gene functions when activated in mice [11].  Female Aedes aegypti mosquitoes mate as a rule before taking a first blood meal [12].  Thus living human blood will be exposed to the piggyBac carried by the mated female.  What would it take to activate the mosquito-borne transposon to infect human blood? No more than an encounter with Baculovirus that could enter through open cuts or sores, or with inhaled dust.  The piggyBac transposon GM construct could wreak havoc in the human genome, causing numerous insertion mutations and other untold, unpredictable damage.

Dengue virus replication cycle and infection

Infection with dengue virus begins when the mosquito takes a blood meal and the virus is introduced into the host. The virus binds to and enters a permissive host cell via receptor-mediated endocytosis. Fusion of viral and endosome-vesicle membranes allows the naked virus to enter the cytoplasm, where its and genome is further stripped of its protein coat. Translation of the protein-coding strand takes place; after which, the virus switches to the synthesis of a negative- (non-coding) strand intermediate that forms a circle and  serves as a template for the production of multiple copies of positive-strand viral RNA (vRNA). Successive rounds of translation produce high levels of viral proteins; the structural protein capsid or core (C), premembrane (prM), and envelope (E) proteins, along with vRNA, are assembled into progeny virions, which are transported through the Golgi compartment and secreted [13].

To conclude

Dengue virus is a growing threat to human health. The problem is compounded by the absence of effective vaccines. However, as we pointed out [1], there are recent safe, effective, and affordable alternatives to controlling the insect vector. In contrast, the current release of terminator male mosquitoes on Cayman Island is a risky strategy. To add insult on injury, there has been no warnings issued to tourists, and most residents on the Island do not appear to have given informed consent to be exposed to the GM male mosquitoes, in blatant violation of human rights with regard to human experimentation.  If the strategy has succeeded, as claimed, the islanders may have been granted temporary respite from the insect vector for Dengue; though replacement mosquito vectors are likely to be blown in from neighbouring islands almost immediately while GM mosquitoes are spreading to them from Cayman Island. The UK government appears not to have exercised appropriate jurisdiction over the human experimentation in its territory; while the scientific community should condemn the use of data to justify the experiment from studies that had not passed peer review.

Article first published 08/12/10

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References

1. Cummins J and Ho MW. Terminator mosquito to control dengue  Science in Society 39, 33- 35, 2008.
2. World Health Organization  Dengue and dengue haemorrhagic fever Fact sheet N°117 March 2009  http://www.who.int/mediacentre/factsheets/fs117/en/print.htm
3. Phuc HK, Andreasen MH, Burton RS, Vass C, Epton MJ, Pape G, Fu G, Condon KC, Scaife S, Donnelly CA, Coleman PG, White-Cooper H, Alphey L. Late-acting dominant lethal genetic systems and mosquito control. BMC Biol 2007, 5, 11.
4. Enserink M. GM mosquito trial alarms opponents, strains ties in Gates-funded project. Science 2010, 330, 1030-1.
5. Alphey L. Engineered sterile mosquitoes for dengue control from lab to field. The American Journal of Tropical Hygiene 2010, 83, Abstract Book.
6. Cummins J. PiggyBac a name to remember. I-SIS Report, 6 March 2001, https://www.i-sis.org.uk/piggybac-pr.php
7. Adelman ZN1, Jasinskiene N1, Peek C1, Travanty EA2, Olson KE2, James AA1. Instability of the piggyBac element in transformed Aedes aegypti. ISMIS 2002. Abstracts of the Fourth International Symposium on Molecular Insect Science. 70pp. Journal of Insect Science, 2, 17.
8. Adelman ZN, Jasinskiene N, Vally KJ, Peek C, Travanty EA, Olson KE, Brown SE, Stephens JL, Knudson DL, Coates CJ, James AA.Formation and loss of large, unstable tandem arrays of the piggyBac transposable element in the yellow fever mosquito, Aedes aegypti. Transgenic Res 2004, 13(5), 411-25.
9. Urschitz J, Kawasumi M, Owens J, Morozumi K, Yamashiro H, Stoytchev I, Marh J, Dee J Kawamoto K, Coates CJ, Kaminski JM, Pelczar P, Yanagimachi R, Moisyadi S. Helper-independent piggyBac plasmids for gene delivery approaches: strategies for avoiding potential genotoxic effects. Proc Natl Acad Sci U S A 2010, 107(18), 8117-22.
10. Galvan DL, Nakazawa Y, Kaja A, Kettlun C, Cooper LJ, Rooney CM, Wilson MH. Genome-wide mapping of PiggyBac transposon integrations in primary human T cells. J Immunother 2009, 32(8), 837-44.
11. Galvan DL, Nakazawa Y, Kaja A, Kettlun C, Cooper LJ, Rooney CM, Wilson MH. Genome-wide mapping of PiggyBac transposon integrations in primary human T cells. J Immunother 2009, 32(8), 837-44.
12. Putnam JL, Scott TW. Blood-feeding behavior of dengue-2 virus-infected Aedes aegypti. Am J Trop Med Hyg 1995, 52(3), 225-7.
13. Friebe P, Harris E. Interplay of RNA elements in the dengue virus 5' and 3' ends required for viral RNA replication .J Virol 2010, 84(12), 6103-18.
14. Girimont TM. Dengue disease. AAOHN J 2010 Oct;58(10):448. doi: 10.3928/08910162-20100928-03