John M. Marshall

Malaria Control with Transgenic Mosquitoes

John Marshall and Charles Taylor review recent advances in the development of transgenic mosquitoes for malaria control.

A Synthetic Gene Drive System for Local, Reversible Modification and Suppression of Insect Populations

Replacement of wild insect populations with genetically modified individuals unable to transmit disease provides a self-perpetuating method of disease prevention but requires a gene drive mechanism to spread these traits to high frequency. Drive mechanisms requiring that transgenes exceed a threshold frequency in order to spread are attractive because they bring about local but not global replacement, and transgenes can be eliminated through dilution of the population with wild-type individuals. These features are likely to be important in many social and regulatory contexts. Here we describe the first creation of a synthetic threshold-dependent gene drive system, designated maternal-effect lethal underdominance (UD(MEL)), in which two maternally expressed toxins, located on separate chromosomes, are each linked with a zygotic antidote able to rescue maternal-effect lethality of the other toxin. We demonstrate threshold-dependent replacement in single- and two-locus configurations in Drosophila. Models suggest that transgene spread can often be limited to local environments. They also show that in a population in which single-locus UD(MEL) has been carried out, repeated release of wild-type males can result in population suppression, a novel method of genetic population manipulation.

Factors Contributing to Urban Malaria Transmission in Sub-Saharan Africa: A Systematic Review

Sub-Saharan Africa suffers by far the greatest malaria burden worldwide and is currently undergoing a profound demographic change, with a growing proportion of its population moving to urban areas. Urbanisation is generally expected to reduce malaria transmission; however the disease still persists in African cities, in some cases at higher levels than in nearby rural areas. Objective. This paper aims to collate and analyse risk factors for urban malaria transmission throughout sub-Saharan Africa and to discuss their implications for control. Methods. A systematic search on malaria and urbanisation was carried out focusing on sub-Saharan Africa. Particular interest was taken in vector breeding sites in urban and periurban areas. Results. A variety of urban vector breeding sites were catalogued, the majority of which were artificial, including urban agriculture, tyre tracks, and ditches. Natural breeding sites varied according to location. Low socioeconomic status was a significant risk factor for malaria, often present in peri-urban areas. A worrying trend was seen in the adaptation of malaria vector species to the urban environment. Urban malaria is highly focused and control programs should reflect this. Conclusion. As urbanisation continues and vector species adapt, continued monitoring and control of urban malaria in sub-Saharan Africa is essential.

The effect of gene drive on containment of transgenic mosquitoes.

Mosquito-borne diseases such as malaria and dengue fever continue to be a major health problem through much of the world. Several new potential approaches to disease control utilize gene drive to spread anti-pathogen genes into the mosquito population. Prior to a release, these projects will require trials in outdoor cages from which transgenic mosquitoes may escape, albeit in small numbers. Most genes introduced in small numbers are very likely to be lost from the environment; however, gene drive mechanisms enhance the invasiveness of introduced genes. Consequently, introduced transgenes may be more likely to persist than ordinary genes following an accidental release. Here, we develop stochastic models to analyze the loss probabilities for several gene drive mechanisms, including homing endonuclease genes, transposable elements, Medea elements, the intracellular bacterium Wolbachia, engineered underdominance genes, and meiotic drive. We find that Medea and Wolbachia present the best compromise between invasiveness and containment for the six gene drive systems currently being considered for the control of mosquito-borne disease.

Public Finance of Private Goods: The Case of College Education

This paper describes a contract theory of public finance of college education that explains why everyone pays for the college education of a lucky minority. The contract provides gambles that families desire. Optimizing the contract determines the taxes paid by all members of society, fees paid by those whose children go to college, the fraction of children who are admitted to college, and the quality of college education. Changes in wealth lead to changes in taxes and admissions, but fees and quality are invariant. The practice of using a cutoff level of precollege achievement to determine admission to college is justified by the theory.

Semele: a killer-male, rescue-female system for suppression and replacement of insect disease vector populations.

Two strategies to control mosquito-borne diseases, such as malaria and dengue fever, are reducing mosquito population sizes or replacing populations with disease-refractory varieties. We propose a genetic system, Semele, which may be used for both. Semele consists of two components: a toxin expressed in transgenic males that either kills or renders infertile wild-type female recipients and an antidote expressed in females that protects them from the effects of the toxin. An all-male release results in population suppression because wild-type females that mate with transgenic males produce no offspring. A release that includes transgenic females results in gene drive since females carrying the allele are favored at high population frequencies. We use simple population genetic models to explore the utility of the Semele system. We find that Semele can spread under a wide range of conditions, all of which require a high introduction frequency. This feature is desirable since transgenic insects released accidentally are unlikely to persist, transgenic insects released intentionally can be spatially confined, and the element can be removed from a population through sustained release of wild-type insects. We examine potential barriers to Semele gene drive and suggest molecular tools that could be used to build the Semele system.

The Cartagena Protocol and genetically modified mosquitoes

To the Editor: The Cartagena Protocol on Biosafety1 is the fundamental document of the United Nations on the responsible use of genetically modified (GM) organisms. Although the protocol applies to GM mosquitoes intended for disease control, its terms were negotiated primarily with concerns over the safety and trade of GM crops in mind. A sub-working group has been assigned by the Ad Hoc Technical Expert Group (AHTEG) on Risk Assessment and Risk Management to develop risk assessment guidelines for GM mosquitoes. Its first guidance document has recently been published following an April 2010 meeting in Ljubljana, Slovenia2 and will be submitted to the Parties of the Protocol at their meeting next month. This is an important document outlining the potential risks of GM mosquitoes to biodiversity and human health; however, several overarching issues were considered to be beyond its scope. In this letter, I outline some of these issues and call for a broader discussion on GM mosquitoes to address their unresolved biosafety concerns. As pointed out in the guidance document, several strategies are being developed to control vector-borne diseases using GM mosquitoes, each requiring its own risk assessment and management considerations. One strategy involves the release of genetically sterile males that, upon mating with wild females, produce unviable offspring, thus resulting in population suppression. The technology for this strategy has already been developed for Aedes aegypti3—the main vector of dengue fever—and its biosafety implications are relatively manageable because transgenes are only expected to persist in the wild for a few generations after release. Other self-limiting strategies are being developed that eliminate transgenes over subsequent generations. Another strategy being developed involves the use of a ‘gene drive system’ to spread disease-refractory genes into mosquito populations, thus rendering entire populations incapable of transmitting diseases4. In support of this strategy, a transposable element has been observed to spread through the worldwide population of Drosophila melanogaster in a few decades. Progress is being made in the development of genes refractory to malaria and dengue fever, and synthetic gene drive systems are being developed for A. aegypti and other mosquito species. If successful, then just a few GM mosquitoes with these constructs would be capable of propagating transgenes over the entire geographical range of a species. Gene drive systems are being developed that are expected to be less capable of spreading between populations; however, this is yet to be shown in an environmental setting. Perhaps the most important issue inadequately addressed by the guidance document is the ability of mosquitoes engineered with gene drive systems to propagate transgenes across national borders in the absence of an international agreement. Regarding gene flow, the document expresses the need to consider “methods to reduce the persistence of the transgene in the environment” in cases where GM mosquitoes have been shown to have adverse effects. As a form of risk management, it also encourages consideration of methods for “ensuring that they [GM mosquitoes] do not establish themselves beyond the intended receiving environment.” However, the acceptability of an open release of GM mosquitoes with gene drive systems that are not shown to have adverse effects is left relatively ambiguous. A strict interpretation of the Cartagena Protocol, on the other hand, suggests that the requirements for a release of GM mosquitoes with invasive gene drive systems may be almost impossible to satisfy. The Advance Informed Agreement (AIA) procedure applies before the first environmental release of GM organisms in another country and grants the importing country the right to request the exporting country to perform a risk assessment at its own expense, part of which is to determine the likelihood of an “unintentional transboundary movement.” If these movements are difficult to prevent, which is certainly the case for GM mosquitoes with invasive gene drive systems, then an environmental release is not allowed. One way around this problem is a multilateral agreement, consistent with the protocol, which would acknowledge that any release of these mosquitoes is intentionally international and has been agreed to by the affected nations. The problem with a multilateral agreement, however, is its scale and feasibility. GM mosquitoes with invasive gene drive systems have the potential to spread transgenes over entire continents. In the context of Zambia’s ban on GM food aid in 2002—during a famine that threatened hundreds of thousands of lives—a unanimous, almost worldwide agreement on GM mosquitoes seems challenging, if not impossible. Despite this, invasive gene drive systems, such as homing endonuclease genes and The A. aegypti mosquito, versions of which have been engineered to have a repressible female-specific flightless/sterile phenotype based on the use of the flight muscle promoter of Actin-4 gene.

Perspectives of people in Mali toward genetically-modified mosquitoes for malaria control

BackgroundGenetically-modified (GM) mosquitoes have been proposed as part of an integrated vector control strategy for malaria control. Public acceptance is essential prior to field trials, particularly since mosquitoes are a vector of human disease and genetically modified organisms (GMOs) face strong scepticism in developed and developing nations. Despite this, in sub-Saharan Africa, where the GM mosquito effort is primarily directed, very little data is available on perspectives to GMOs. Here, results are presented of a qualitative survey of public attitudes to GM mosquitoes for malaria control in rural and urban areas of Mali, West Africa between the months of October 2008 and June 2009.MethodsThe sample consisted of 80 individuals - 30 living in rural communities, 30 living in urban suburbs of Bamako, and 20 Western-trained and traditional health professionals working in Bamako and Bandiagara. Questions were asked about the cause of malaria, heredity and selective breeding. This led to questions about genetic alterations, and acceptable conditions for a release of pest-resistant GM corn and malaria-refractory GM mosquitoes. Finally, participants were asked about the decision-making process in their community. Interviews were transcribed and responses were categorized according to general themes.ResultsMost participants cited mosquitoes as one of several causes of malaria. The concept of the gene was not widely understood; however selective breeding was understood, allowing limited communication of the concept of genetic modification. Participants were open to a release of pest-resistant GM corn, often wanting to conduct a trial themselves. The concept of a trial was reapplied to GM mosquitoes, although less frequently. Participants wanted to see evidence that GM mosquitoes can reduce malaria prevalence without negative consequences for human health and the environment. For several participants, a mosquito control programme was preferred; however a transgenic release that satisfied certain requirements was usually acceptable.ConclusionsAlthough there were some dissenters, the majority of participants were pragmatic towards a release of GM mosquitoes. An array of social and cultural issues associated with malaria, mosquitoes and genetic engineering became apparent. If these can be successfully addressed, then social acceptance among the populations surveyed seems promising.

Overcoming evolved resistance to population-suppressing homing-based gene drives

The recent development of a CRISPR-Cas9-based homing system for the suppression of Anopheles gambiae is encouraging; however, with current designs, the slow emergence of homing-resistant alleles is expected to result in suppressed populations rapidly rebounding, as homing-resistant alleles have a significant fitness advantage over functional, population-suppressing homing alleles. To explore this concern, we develop a mathematical model to estimate tolerable rates of homing-resistant allele generation to suppress a wild population of a given size. Our results suggest that, to achieve meaningful population suppression, tolerable rates of resistance allele generation are orders of magnitude smaller than those observed for current designs for CRISPR-Cas9-based homing systems. To remedy this, we theoretically explore a homing system architecture in which guide RNAs (gRNAs) are multiplexed, increasing the effective homing rate and decreasing the effective resistant allele generation rate. Modeling results suggest that the size of the population that can be suppressed increases exponentially with the number of multiplexed gRNAs and that, with four multiplexed gRNAs, a mosquito species could potentially be suppressed on a continental scale. We also demonstrate successful proof-of-principle use of multiplexed ribozyme flanked gRNAs to induce mutations in vivo in Drosophila melanogaster – a strategy that could readily be adapted to engineer stable, homing-based drives in relevant organisms.

Confinement of gene drive systems to local populations: a comparative analysis.

Mosquito-borne diseases such as malaria and dengue fever pose a major health problem through much of the world. One approach to disease prevention involves the use of selfish genetic elements to drive disease-refractory genes into wild mosquito populations. Recently engineered synthetic drive systems have provided encouragement for this strategy; but at the same time have been greeted with caution over the concern that transgenes may spread into countries and communities without their consent. Consequently, there is also interest in gene drive systems that, while strong enough to bring about local population replacement, are unable to establish themselves beyond a partially isolated release site, at least during the testing phase. Here, we develop simple deterministic and stochastic models to compare the confinement properties of a variety of gene drive systems. Our results highlight several systems with desirable features for confinement-a high migration rate required to become established in neighboring populations, and low-frequency persistence in neighboring populations for moderate migration rates. Single-allele underdominance and single-locus engineered underdominance have the strongest confinement properties, but are difficult to engineer and require a high introduction frequency, respectively. Toxin-antidote systems such as Semele, Merea and two-locus engineered underdominance show promising confinement properties and require lower introduction frequencies. Killer-rescue is self-limiting in time, but is able to disperse to significant levels in neighboring populations. We discuss the significance of these results in the context of a phased release of transgenic mosquitoes, and the need for characterization of local ecology prior to a release.