Gene drive as a tool to fight mosquito-borne diseases – GDaMo

We use the Golden Gate Cloning technology to assemble complex plasmid constructs, comprising the many modules that are necessary for gene drive activity, for anti-pathogen activity, and for the tracking of the genetic modifications in the obtained mosquito lines. After the process of transgenesis, we breed the transgenic mosquito lines over many generations and analyse the dynamics of transgenes (notably using flow cytometry on live neonate larvae), and make use of infection assays to evaluate their resistance.

We have established an anti-Plasmodium gene drive (GD) system in the Anopheles mosquito (Green et al., eLife 2023). The driving part of this GD is integrated into the Saglin locus and targets both the Saglin and Lipophorin genes. This GD is capable of invading an Anopheles population in which a modified version of lipophorin has been previously introduced, blocking Plasmodium transmission and adding to the transmission reduction due to the loss of Saglin function. By monitoring the dynamics of the transgenes over 26 generations, we verified that the two genetic modifications spread rapidly and in a mutually dependent manner within a mosquito population, reducing vector competence. GD resistance mutations eventually accumulate, which in the long term would cause both transgenes to disappear, constituting a form of reversibility of the intervention.

In the Aedes aegypti mosquito, vector of dengue, Zika and chikungunya, we have established a toxin-antidote system in the form of a modification of the lipophorin gene sequence (antidote), rendering it insensitive to the components of the CRISPR-Cas9 system (toxin) programmed to attack the wild-type version of lipophorin and expressed from a second transgene. These components can now be combined into a single locus, which will also carry an antiviral factor. Together, these components will constitute a toxin-antidote type GD with a high invasion threshold, which should enable local modification of mosquito populations (spatial confinement of transgenes) rendering them resistant to pathogenic viruses.

We will continue to optimize the combined gene drive system to reduce the ability of Anopheles mosquitoes to transmit malaria, and develop additional anti-Plasmodium transgenes that can be combined with those we have already characterized. We will study the ability of Plasmodium to evolve to circumvent these barriers raised against them within the mosquito, in order to assess the risk of resistance and the sustainability of interventions based on these transgenes.

In the Aedes mosquito, we will finalize our gene drive system against viruses by assembling the already characterized components into a single locus. We will measure the dynamics of the synthetic locus thus constructed and its antiviral efficacy.

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Mosquito vectors of human disease are still responsible for the death of about 750,000 people annually. Genetic resistance to insecticides is spreading in mosquito populations, new arthropod-borne viral diseases emerge continuously and drugs against malaria stimulate the emergence of drug-resistant parasites. In our fight against mosquito-borne disease, the gene drive technology shows great promise to complement existing vector control methods. Gene drives are transgenic constructs designed to invade populations of a target species, based on the activity of molecular scissors such as the CRISPR/Cas9 system. Gene drive could be deployed either for population suppression, which may cause a loss in biodiversity and ecosystem imbalance, or for genetic modification at the scale of a species. In confined laboratory conditions, we will develop gene drive constructs carrying anti-pathogen molecules to render Anopheles gambiae and Aedes aegypti mosquitoes unable to transmit malaria parasites and viruses (dengue, Zika). We will optimize and test several innovative gene drive approaches, with particular attention to avoid the emergence of drive-refractory mosquitoes: multiplex drives; anti-Plasmodium dual-effect drive; drive-or-die constructs; indirect drives. We will monitor the efficiency of each of these strategies in caged mosquitoes, the possible undesired effects arising from on and off-target mutations induced by the drive, develop “recall strains” able to block the drive, and test the constructs’ propensity for horizontal transfer towards other insect species and microorganisms. Project partners have complementary expertise in Anopheles and Aedes biology, in gene engineering and in mosquito infection assays with human pathogens. This project aims both to optimize gene drive and to address some of the problems and concerns raised by this research field. We hope it will foster the emergence of alternative approaches to insecticide-based control interventions, which are reaching their limits.