Malaria remains a major public health concern, with many African nations being far from meeting their malaria elimination targets4,5. Vector control methods including indoor residual spraying and long-lasting insecticide-treated bed nets have played a pivotal role in reducing malaria incidence, but the emergence of insecticide-resistant mosquitoes has impeded further progress6. In addition, Africa’s rapidly growing population and persistent malaria receptivity make these interventions increasingly unsustainable as standalone solutions. This highlights the urgent need for innovative, self-sustaining and cost-effective technologies to complement existing efforts in malaria elimination. Gene drive technology, which enables the biased inheritance of selected traits and can spread through populations at rates exceeding those predicted by Mendelian genetics, has emerged as a promising new paradigm7,8.
Gene drive can offer a transformative solution for malaria elimination by spreading genetic modifications that can either suppress mosquito populations or render them unable to transmit the disease2,9,10. Our work focuses on the latter approach known as mosquito population modification or replacement, whereby antiparasitic effectors introduced into the mosquito genome are spread to fixation within populations using a Cas9 endonuclease-based synthetic gene drive. In our design, the transmission-blocking effector and gene drive functions are separated into distinct genetic traits and strains3,11,12,13. This separation offers several advantages: it allows the development, testing and optimization of effector constructs in endemic settings independently of a full gene drive system; it facilitates rigorous risk assessment and community engagement before introducing self-propagating elements and it provides a safer, more modular pathway towards deployment12. Crucially, evaluating non-autonomous effector strains helps address elevated regulatory and containment requirements associated with autonomous gene drive systems.
Genetic modification of mosquitoes to reduce their vectorial capacity was first attempted more than two decades ago, and dozens of transgenic strains have been described in the literature to date9,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30. However, no effector has ever been evaluated against parasites other than laboratory strains many of which were established in the early 1980s31. For this reason, their propensity to block the transmission of genetically diverse Plasmodium isolates now in circulation is unknown.
We previously demonstrated the efficacy of one such A. gambiae effector modification in inhibiting the NF54 strain of laboratory-cultured P. falciparum. This modification, termed MM-CP, involves two antimicrobial peptides, magainin 2 from the African clawed frog and melittin from the European honeybee32, integrated into and expressed from within the endogenous zinc carboxypeptidase A1 gene (CP)33. This minimal genetic modification that harbours no fluorescent markers interferes with oocyst development causing a significant delay in the release of infectious sporozoites. It also reduces the lifespan of homozygous female mosquitoes, further minimizing their potential to transmit malaria. Predictive models suggest that gene-drive-mediated population-wide propagation of MM-CP could disrupt disease transmission across various settings, offering promise for malaria elimination even in scenarios in which resistance to the effector or the drive eventually emerge. Here we adapted this technology for an African context to evaluate its ability to suppress P. falciparum parasites naturally circulating among humans.
The implementation of gene drive technologies in malaria-endemic regions faces substantial challenges, including limited access to appropriate containment infrastructure, regulatory uncertainty, insufficient local capacity for genetic engineering and biosafety, and the imperative for community trust and public transparency. To enable our work, we developed an integrated Modular Portable Laboratory and Containment Level 3 (MPL/CL3) insectary facility, specifically designed for generating, housing and studying genetically modified mosquitoes within an African context (Fig. 1a). The MPL/CL3 was designed to address some of these constraints by offering a high-security and standardized facility tailored to local environmental and regulatory conditions. It incorporates climate and illumination control systems, rearing chambers, microbiologically safety cabinets, water management and waste disposal systems, an autoclaving unit and a fully equipped laboratory. The facility was constructed within two intermodal shipping containers in Spain and transported and installed at the Bagamoyo campus of the Ifakara Health Institute (IHI) in Tanzania (Fig. 1b). By embedding cutting-edge vector biology capacity within endemic settings, the MPL/CL3 supported local research leadership, regulatory readiness and public engagement, laying essential groundwork for responsible development and evaluation of gene drive technologies. Detailed specifications and technical plans are presented in the Methods and Supplementary Note. All protocols involving the generation and study of transgenic mosquitoes were reviewed and approved by the relevant institutional and national regulatory authorities in Tanzania.
Fig. 1: Infrastructure capacity building and malaria surveillance sites in northeastern Tanzania. a, Integrated MPL/CL3 facility. Architectural design plans for the (left) and a detailed view of the integrated laboratory and insectary container unit (right) are shown. The laboratory comprises a lobby, an incubator room for mosquito husbandry, a molecular biology laboratory and a dedicated space for P. falciparum DMFAs and housing of infected mosquitoes. The second container unit houses systems that regulate and maintain optimal environmental conditions, including a negative pressure system for biosecurity, water purification and waste treatment. An external electricity generator supports these operations. b, Field sites for parasitological surveys and gametocyte carrier recruitment. Locations of villages in the Pwani region where parasitological surveys were conducted in children are shown in relation to the IHI Bagamoyo campus (housing the MPL/CL3 facility), the capital Dodoma, the major port city Dar es Salaam and the town of Chalinze, where meteorological data were recorded. The map is modified to highlight sites mentioned in the paper. Tanzania road map in b adapted from OnTheWorldMap.com (https://ontheworldmap.com/tanzania/tanzania-road-map.html). Full size image
The first A. gambiae transgenic line developed onsite within the MPL/CL3, named zpg-CC, was designed to streamline all transgenesis processes by expressing both Cre recombinase and Cas9 endonuclease under the control of the zero-population growth (zpg) gene promoter. This dual helper strain enables the efficient removal of sequences such as transgenesis markers flanked by loxP sites and establishment of transgene homozygosity through homing. The initial development and characterization of the zpg-CC line were conducted at Imperial, before the line was recreated in Tanzania.
The zpg-CC construct includes a dominant DsRed transgenesis marker, integrated into the kynurenine hydroxylase (kh) gene locus (Extended Data Fig. 1a). Disruption of both copies of the gene results in white-eyed mosquitoes, serving as a recessive phenotypic marker. Although the zpg-CC helper line was robust and fertile, it showed reduced overall fitness, probably due to the disruption of the kh locus and/or the effects of germline-specific or leaky expression of both Cre and Cas9. Compared with wild-type (wt) females, sugar-fed transgenic homozygous zpg-CC females showed a small decline in survival over time (Extended Data Fig. 1b), consistent with previous observations in other mosquitoes34,35. They also laid significantly fewer eggs after blood feeding, with a lower proportion hatching, indicating a reduction in reproductive fitness (Extended Data Fig. 1c).
To assess the efficiency of the zpg-CC helper line in inducing homing when combined with a non-autonomously driveable transgene expressing guide RNA (gRNA), we crossed heterozygous zpg-CC males with females of a previously generated CP knockout (CP-KO) line that harbour a green fluorescent protein (GFP) expression cassette and a gRNA expression module inserted within and targeting the CP gene36 (Extended Data Fig. 1a). Heterozygous offspring expressing both GFP and DsRed were sib-mated, and the resulting larvae were screened for green fluorescence. All 623 larvae screened were GFP positive, compared with the 75% expected from a Mendelian intercross of hemizygotes. This indicates 100% Cas9-mediated homing, induced by Cas9 provided by the zpg-CC helper line (Extended Data Fig. 1d).
Next, we assessed the capacity of the zpg-CC helper line to excise a loxP-flanked GFP expression cassette through the expression of Cre recombinase. As a tester line we used the zpg-Cas9GFP strain, in which a Cas9 coding sequence was inserted within the zpg gene to encode Cas9 linked to the zpg C terminus through an E2A ribosome-skipping peptide sequence. An intron harbouring the excisable GFP expression cassette flanked by loxP sites and a gRNA module is located within the E2A sequence (Extended Data Fig. 1a). We crossed zpg-Cas9GFP males with heterozygous zpg-CC females, selecting GFP and DsRed positive males for subsequent crosses with wt females (Extended Data Fig. 1e). Among 417 offspring larvae, only 13 showed green fluorescence, indicating efficient Cre-mediated excision of the GFP cassette (97%).
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