Engineering the control of mosquito-borne infectious diseases

Abstract

Recent advances in genetic engineering are bringing new promise for controlling mosquito populations that transmit deadly pathogens. Here we discuss past and current efforts to engineer mosquito strains that are refractory to disease transmission or are suitable for suppressing wild disease-transmitting populations.

Figure 1

Methods for the genetic control of vector populations. (a) Population suppression can be achieved by releasing large numbers of males that render their wild female mates incapable of having viable progeny. This includes releasing either males that are sterile and produce no progeny at all (as in sterile insect technique (SIT)) [15] or males that pass on lethal transgenes to the next generation, producing progeny that die before they can transmit disease (as in the release of insects carrying dominant lethals, RIDL) [16]. For SIT strategies, multiple releases of a large excess (5x to 10x) of sterile males relative to the target population are normally carried out over large areas. (b) Population replacement occurs when traits carried by a small number of engineered mosquitoes replace traits that naturally exist in field populations [17]. The desired engineered trait - for instance, an anti-pathogen gene that renders mosquitoes refractory to disease transmission - is driven to fixation in the field population using a genetic drive (as described in Figure 2h).

Figure 2

Current and future genetic engineering technologies for vector control. (a) First-generation technologies make use of transposable elements to insert genetic cargo randomly into the genome. The transposable element is mobilized by a transposase enzyme produced by another plasmid, which recognizes and cleaves the terminal repeats (TR) of the transposon cassette and mediates insertion of the transposable element into the genome. Insertion is visualized using selectable markers such as the green fluorescent protein (GFP) [19]. (b) Mosquitoes can be engineered to carry anti-pathogenic effector genes that reduce the pathogen load [21-31]. In the figure, the effector gene blocks Plasmodium ookinete invasion of the midgut epithelium, preventing oocyst development. (c) Schematic of the RIDL system currently used for suppression of Aedes aegypti populations [16]. In the presence of tetracycline, expression of the tetracycline transactivator (tTA) is repressed. In the absence of tetracycline, tTA binds to the tetracycline-responsive element (tRE) and drives its own expression in a positive feedback loop that leads to the accumulation of toxic levels of tTA. The progeny of released males carrying this transgene are not viable. Other combinations of inducible systems and toxic genes can be used in place of tTA and tRE to achieve population suppression. (d) Second generation technologies include HEGs, ZFNs, TALENs and CRISPR/Cas9 [11-13,32,33]. These technologies facilitate double-stranded DNA breaks in the genome at desired loci. (e) HEGs, TALENs and ZFNs have been used in Ae. aegypti and Anopheles gambiae to generate null mutants [11-13], including eye color mutants [11]. (f) ZFNs have been used to generate site-specific knock-ins of exogenous sequences in Ae. aegypti [34]. The figure illustrates a possible application for knock-in technology, which would enable scientists to fuse protein domains to the end of endogenous genes. These domains include those encoding fluorescent proteins or epitope tags, such as an HA tag (shown). (g) Sex distorter strains make use of an HEG, I-PpoI, to destroy sperm carrying an X chromosome (X-shredder), producing male-only populations. When mated to wild-type females, transgenic males sire only sons, potentially leading to population suppression [35]. (h) Gene drives are genetic elements that are inherited in a non-Mendelian fashion and can spread through populations. Gene drives using HEGs have been successfully developed to drive through laboratory mosquito populations [36], whereas evolutionarily stable drives enabled by CRISPR/Cas9 have been proposed [37].

Figure 3

Challenges for the field release of transgenic mosquitoes. This scheme summarizes the ecological, behavioral and regulatory issues faced by disease control programs based on the release of genetically modified mosquitoes. Ecological requirements are shown in green, behavioral requirements in orange, while regulatory issues are presented in blue. Light-grey sections highlight operational tools that may be used to comply with the requirements. Behavioral requirements include key fitness parameters such as the dispersal ability and mating competitiveness of released males, and can be tested in large laboratory cage trials and then in semi-field settings to select the mosquito strains with the greatest probability of success. Ecological hurdles comprise heterogeneity in the genetics, behavior and natural habitats of vector species (biodiversity), and possible unintended side-effects on non-target species or on the ecosystem. Monitoring of these effects must be constantly in progress in the release phase. The risks, safety and specificity of the engineered strains need to be evaluated by appropriate regulatory agencies, and early public engagement is a priority.