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Considerations for first field trials of low-threshold gene drive for malaria vector control

Abstract

Sustainable reductions in African malaria transmission require innovative tools for mosquito control. One proposal involves the use of low-threshold gene drive in Anopheles vector species, where a ‘causal pathway’ would be initiated by (i) the release of a gene drive system in target mosquito vector species, leading to (ii) its transmission to subsequent generations, (iii) its increase in frequency and spread in target mosquito populations, (iv) its simultaneous propagation of a linked genetic trait aimed at reducing vectorial capacity for Plasmodium, and (v) reduced vectorial capacity for parasites in target mosquito populations as the gene drive system reaches fixation in target mosquito populations, causing (vi) decreased malaria incidence and prevalence. Here the scope, objectives, trial design elements, and approaches to monitoring for initial field releases of such gene dive systems are considered, informed by the successful implementation of field trials of biological control agents, as well as other vector control tools, including insecticides, Wolbachia, larvicides, and attractive-toxic sugar bait systems. Specific research questions to be addressed in initial gene drive field trials are identified, and adaptive trial design is explored as a potentially constructive and flexible approach to facilitate testing of the causal pathway. A fundamental question for decision-makers for the first field trials will be whether there should be a selective focus on earlier points of the pathway, such as genetic efficacy via measurement of the increase in frequency and spread of the gene drive system in target populations, or on wider interrogation of the entire pathway including entomological and epidemiological efficacy. How and when epidemiological efficacy will eventually be assessed will be an essential consideration before decisions on any field trial protocols are finalized and implemented, regardless of whether initial field trials focus exclusively on the measurement of genetic efficacy, or on broader aspects of the causal pathway. Statistical and modelling tools are currently under active development and will inform such decisions on initial trial design, locations, and endpoints. Collectively, the considerations here advance the realization of developer ambitions for the first field trials of low-threshold gene drive for malaria vector control within the next 5 years.

Background

Insecticide-treated bed nets (ITNs) and indoor residual insecticide spraying (IRS) are currently the main tools for malaria vector control, which mainly act on vectorial capacity by reducing the density and longevity of adult female mosquitoes [1, 2]. While new insecticides, spatial repellents, housing modifications, and mosquito traps are in the research and development pipeline, both the WHO and African Union have recognized that these will be insufficient to meet the challenges of reducing the malaria burden and new transformational tools will be required along with additional deployment of existing interventions [3, 4]. Among such tools, genetic approaches, including gene drive systems that target the mosquitoes that transmit malaria parasites are currently being developed and evaluated for potential use in the field.

Gene drive systems promote the biased inheritance of specific genes from one generation to the next [5,6,7,8]. While several disparate mechanisms exist, the biased inheritance of the most well-studied type is achieved via a process known as ‘homing’ that exploits CRISPR biology [8]. Here the gene drive system is (i) inserted at a specific genomic target locus on one of a pair of homologous chromosomes and encodes both (ii) the Cas endonuclease under control of a promoter with germline activity and (iii) a guide RNA that targets the specific genomic locus into which the gene drive system is inserted and homes. In germline cells, the Cas/gRNA complex causes a double-strand break in the genomic target locus on the homologous chromosome where the gene drive system is absent [7,8,9]. Homology-directed repair uses the chromosome encoding the gene drive system as a template to repair that double-stranded break and in doing so, copies the gene drive system from the intact chromosome onto the cleaved homologous chromosome.

The net result of this homing reaction is that both copies of homologous chromosomes in the germline contain the gene drive system, so that most gametes, and consequently most progeny, will possess a copy of gene drive system at the genomic target site. Importantly, homing of a gene drive system also can be associated with the introduction into mosquito target populations of a genetic trait that reduces vectorial capacity, by either reducing the density of Anopheles females in the case of “population suppression”, or impeding their ability to transmit Plasmodium parasites, in “population modification”, also called “population replacement”. Thus, genetic efficacy is used to refer to the efficiency in the field with which the gene drive system can increase in frequency, or proportion, while simultaneously introducing genetic traits that can reduce vectorial capacity, in target mosquito populations at release locations, as well as spread into target mosquito populations at distal locations [10].

While disparate gene drive systems can be designed to have a range of levels of increase in frequency, spread and persistence in target populations, it is the self-sustaining, non-localizing characteristics inherent in “low threshold” gene drive systems that would be expected to require minimal numbers of mosquitoes to be released for achieving efficacy in the field, making them particularly attractive for malaria control. Low-threshold gene drive systems are designed to progressively increase in frequency and spread indefinitely, in every generation post-release, and eventually persist at stable levels (Fig. 1) [6, 10, 11].

Fig. 1
figure 1

Conceptual diagram illustrating temporal and spatial dynamics of gene drive systems. A1 Gene drive mosquitoes are released at specific release locations (black dots) and individual mosquitoes disperse to different habitats [12, 110, 167]. A2 Over time and multiple generations, the gene drive progressively increases in frequency, (measured as a percent, or proportion, of gene drive mosquitoes from the total mosquito population) and, aided by the dispersal of individual mosquitoes to different habitats, spreads to other individuals through interbreeding in target populations (white arrow) [12]. A3 The gene drive persists in this area (dome within light-blue dotted borders) and continues to spread further through target populations (white arrow). B1 In the case of population suppression gene drive, the gene drive system is released in mosquitoes which disperse from release locations. B2 The gene drive system increases in frequency at release locations, observed as an increase in frequency of mosquitoes heterozygous for the gene drive system. The gene drive also spreads to distal target populations, increasing in frequency as it does so. Increasing numbers of mosquitoes that are heterozygous for the gene drive leads to increasing chances of heterozygous males mating with heterozygous females, thus increasing the number of female progeny that are homozygous for the gene drive system, and thus sterile. B3 This leads to a progressive reduction in the density of the target mosquito populations, propagating out over time from release locations, with the spectrum of navy to sky blue indicating high to low population densities in the illustration. These progressive reductions in vector numbers would correlate with progressive reductions in the incidence of malaria, with the spectrum of navy to sky blue indicating high to low malaria incidence in the illustration, and the progressive nature of these effects indicated by grey arrows. No direct change in the prevalence and intensity of infectious Plasmodium sporozoites in mosquitoes would be anticipated initially, although over time with anticipated reduced prevalence of malaria in humans, sporozoite rates in mosquitoes would be reduced. The frequency of the gene drive system in the mosquito population should be unaffected by its suppression, unless the population was to be eliminated completely. C1 For population modification gene drive, the gene drive system is released in mosquitoes which disperse from release locations. C2 The gene drive system increases in frequency. C3 This would be associated with progressive decreases in sporozoite rates in target populations of mosquito, leading to progressive reductions in the incidence of malaria, without any anticipated changes in the densities of mosquito target populations

As low-threshold gene drive systems reach fixation in target populations, they are intended to meet the prerequisites for a novel vector control tool by: (i) reducing the vectorial capacity of target mosquito populations for Plasmodium transmission, referred to here as entomological efficacy, and (ii) reducing the incidence and prevalence of malaria in humans, referred to here as epidemiological efficacy, while being effective over wide geographic areas, acting at low cost, and not requiring human behavioural change. Low-threshold gene drive systems could, therefore, be deployed to help reduce transmission rates in high transmission areas, particularly in areas where other more human-intensive delivery methods are challenging, or in low transmission areas to help accelerate malaria elimination, or to help prevent reintroduction and transmission in areas that have been declared malaria-free [4].

Over 2022 and 2023, the GeneConvene Global Collaborative of the Foundation for the National Institutes of Health (FNIH) assembled a group of developers of low-threshold gene drive systems for malaria vector control that are considered to be at the closest stages of readiness for proposals of field-testing. The group considered some of the key challenges, specific efficacy and risk research questions, knowledge gaps, and potential solutions that would apply to initial field release trials, building on the ‘Guidance framework for testing genetically modified mosquitoes’ (GMMs) of the WHO [12]. The term “trial” was used in its broadest context here, analogous to the range of designs typically found in pharmaceutical development programmes, from Phase 0 trials assessing pharmacodynamic and pharmacokinetic endpoints or Phase I dose-finding trials, both of which do not involve controls, to Phase II trials assessing the intervention compared to a placebo control group, through to Phase III trials often involving comparison with the standard of care [13]. Informed by a broad range of consultations with other experts in entomology, epidemiology, and vector control field trials, the outcomes of those deliberations are presented here.

Trial objectives

For a low threshold gene drive system to be successful as a malaria vector control tool in the field, a ‘causal pathway’ would be initiated by (i) the release of a gene drive system in target mosquito vector species, leading to (ii) its transmission to subsequent generations, (iii) its increase in frequency and spread in target mosquito populations, (iv) its simultaneous propagation of a linked genetic trait aimed at reducing vectorial capacity for Plasmodium, and (v) reduced vectorial capacity for Plasmodium in target mosquito populations as it reaches fixation in target mosquito populations, resulting in (v) decreased malaria incidence and prevalence (Fig. 2). Therefore, assessments of the efficacy of some or all of this causal pathway will be key objectives for initial field trials of low-threshold gene drive. Choices on objectives of those initial field trials will fundamentally inform their design so that explicit decisions will be required a priori on whether the focus and design for these initial field release trials should rest exclusively on assessments of genetic, entomological, or epidemiological efficacy, or on combinations thereof [12, 14,15,16,17,18]. In the WHO guidance framework for testing GMMs, it is envisaged that both genetic efficacy and entomological efficacy would be tested in Phase 2, following the successful completion of Phase 1 laboratory, insectary, and modelling studies [12]. To maintain consistency with WHO terminology, assessment of genetic efficacy as proposed here could occur in Phase 2 “A” of initial field trials, entomological efficacy at Phase 2 “B”, and epidemiological efficacy at Phase 3 (see Box 1 and Fig. 2).

Fig. 2
figure 2

Potential design features for initial field trials of low-threshold gene drive systems. There are three potential levels of efficacy, which could be assessed separately or in different combinations in initial field trials. Where genetic efficacy (WHO Phase 2 “A”; [12]) would be the sole efficacy objective of initial field trials, this could potentially be assessed in observational studies without control locations, given the pre-release absence of the gene drive system from wild target mosquito populations, provided potentially confounding factors such as rainfall were recorded. Where the objectives of an initial field trial were also to assess entomological (WHO Phase 2 “B”) and epidemiological efficacy (WHO Phase 3), cRCTs would provide the most robust estimates of entomological and epidemiological efficacy with least opportunity for introduction of bias from confounding factors. Genetic efficacy of a low threshold gene drive system involves its increase in frequency at, and spread from, the release location in target populations. This could be measured using a variety of endpoints and field methodologies as outlined. Entomological efficacy could be established by measuring the impact of the low threshold gene drive on vectorial capacity using highlighted endpoints and methodology. Epidemiological efficacy could be measured as impact on rate of infection or malaria incidence and prevalence, as indicated. The range of endpoints chosen for initial field trials, whether addressing genetic, entomological, or epidemiological efficacy, will depend on power calculations for specific field methods underpinning them, as well as operational and cost considerations. In addition to efficacy assessments, considerations of the design of initial field trials may consider operational issues, the potential to assess potential safety endpoints, in addition to capturing data and information before and during trials on potential confounding factors that could impact efficacy assessments. Furthermore, considerations of the scope of efficacy measurements will also need to consider the higher levels of oversight and governance that accompany epidemiological efficacy assessment as clinical trials

Trial design

Key considerations for the trial design will include whether or not to include both release locations, where the gene drive system would be released, and control locations, where it would be absent, preferably over the entire course of the study [12, 68]. For example, in longitudinal trials of entomological efficacy where data collected at release locations before and after release of the gene drive system, confounding factors such as changes in rainfall or malaria transmission could augment or obscure before-and-after comparisons on the impact of the gene drive system on target mosquito populations [15]. In that regard, and depending on its objectives, a ‘controlled before-and-after’, or ‘pre-post control group’, trial design could be considered for such initial gene drive trials. Here, data are collected before and after the introduction of the intervention in both the experimental group receiving the intervention and control group that does not receive the intervention [15]. Such a design has been used to assess integrated malaria vector control using ITNs and microbial larvicides in Western Kenya [15, 69].

Cluster randomized controlled trials (cRCTs) [18], in which households, villages, or broader geographical areas (“clusters”) are randomly assigned either to the intervention or to the control, are considered to provide the most robust estimates of entomological and epidemiological efficacy with the least opportunity for the introduction of bias from confounding factors, although technical or ethical considerations in some vector control trials may necessitate the use of alterative designs [12, 15, 16, 18, 70, 71]. cRCTs have previously been used in numerous vector control field trials [15, 16], such as those involving ITNs [72] and other insecticide-based interventions [15, 16, 73,74,75] including eave tubes [76], or spatial repellants [77,78,79,80], ivermectin mass drug administration [81, 82], the wMel strain of Wolbachia pipientis (Rickettsiales; Anaplasmataceae) [83,84,85], and attractive-toxin sugar bait traps [86,87,88].

The key elements to influence the power of a cRCT are (i) the number of clusters, (ii) the number of individuals within each cluster, (iii) the magnitude of the baseline measurement endpoint, (iv) the minimum difference in measurement endpoint between control and intervention arms for which the trial is powered, (v) the variation between clusters in the measurement endpoint, and (vi) the necessary separation distance between clusters to minimize “spillover effects” of the intervention between study arms [15, 18, 50]. Because primary outcomes in vector control trials are often based on epidemiological endpoints, they have typically been designed with statistical power to detect epidemiological, rather than entomological, impacts [69, 76, 86,87,88,89].

Where genetic efficacy would be the sole efficacy objective of initial field trials, this could potentially be assessed in observational studies without control locations, given the pre-release absence of the gene drive system from wild target mosquito populations, provided potentially confounding factors such as rainfall were also recorded [15]. Therefore, at the simplest level, initial field trials could involve release locations as “point sources” so that measurement endpoints would comprise the rate of increase in frequency of the gene drive system in target mosquito populations, as well as its linked traits affecting vectorial capacity, and the rate and variability of its spread to distal locations (Fig. 1).

Unlike current interventions, one of the attractions of the low threshold gene drive as a malaria vector control tool is that it is designed to spread and persist indefinitely in target populations. Hence, once these initial genetic efficacy trials were complete, it could be anticipated that the gene drive system would continue to spread and persist indefinitely in target mosquito populations. Given these properties, the released gene drive system could eventually reach control locations, effectively spilling over and “converting” those initial control locations to “passive” release locations in subsequent stages of the trial [18]. Consequently, where only genetic efficacy was to be assessed in initial field trials, locations of subsequent trials assessing entomological or epidemiological efficacy would need to be sufficiently distant to initial release locations to avoid the risk of spillover.

Where the objectives of an initial field trial were to assess entomological and epidemiological efficacy subsequent to genetic efficacy, at least two different strategies could be considered. In the first, the outcomes of the initial genetic efficacy field trial could inform the design of a later, and entirely separate, cRCTs measuring entomological and epidemiological endpoints, whereby the initial study locations would have to be sufficiently distantly located from the study locations of the subsequent trial to avoid the potential for spillover effects from the first trial to the second [18]. While the genetic efficacy outcomes from the first trial could differ to some degree from genetic efficacy outcomes in the subsequent trial, they would still provide an important scale of effect for later trial design.

In the second strategy for trials measuring genetic, and entomological, and epidemiological efficacy, the initial field trial could be designed as a single, integrated cRCT, which from the inception of its the genetic efficacy phase, would involve random allocation of clusters as release or control locations that would subsequently be used for entomological and epidemiological endpoints. In this case, a key constraint in a cRCT would be that all study locations would need to form part of a framework of random allocation [18]. To both accommodate the potential uncertainty in outcomes from the initial genetic efficacy phase of the trial and to allow optimization of the final design of subsequent entomological and epidemiological efficacy phases, such a trial could be adaptively designed [12, 68] to incorporate some flexibility in the duration of the genetic efficacy phase of the trial, albeit potentially increasing the risk of spillover. Therefore, in this kind of strategy, subsequent to genetic efficacy measurements both entomological and epidemiological endpoints would likely be assessed in parallel, with the entire field trial most likely being statistically powered for epidemiological primary endpoints [15].

Transitions in the gene drive system status of control locations to release locations could potentially be accommodated by a type of cRCT design known as a stepped-wedge trial, in which an intervention is progressively rolled out across all trial locations or clusters [15, 18]. For example, a stepped-wedge trial design was used to assess the epidemiological impact on malaria of solar-powered odour-baited mosquito trapping systems in Kenya [90] and has been explored in the design of dengue vector control trials [91], although in the case of a low-threshold gene drive system, its spread from experimental to control locations would not formally be a randomized process. A so-called “fried egg” design has been employed in some cRCTs to allow primary outcomes to be measured within a core area from each cluster to limit spillover impacts from the intervention into controls [18, 88]. A related approach could employ sufficient distance, or “buffer zones” (Fig. 4), between release and control locations to limit the possibility that the gene drive system would spread to control locations over the course of the initial field trial, provided that such control locations retained similarity to release locations for entomological, environmental, ecological, and epidemiological attributes, which could be ensured through restricted randomization [18, 50]. Trials can also be designed to account for spillover between release and control locations via statistical design elements in addition to the use of buffer zones [14, 15, 18, 84, 85]. A recent modelling study has shown that results of seminal Wolbachia cRCTs demonstrating significant control of dengue transmission may have actually underestimated the true efficacy of the intervention because of three spillover effects caused by (1) human movement, (2) mosquito movement, and (3) disease transmission coupling between treatment and control clusters, indicating the importance of modelling in design and interpretation of future trials of low threshold gene drive for malaria vector control [92]. Additionally, statistical analyses of data collections from locations along gradients of changing frequency of the gene drive system, from release locations through buffer zones to control locations, could also be highly informative in testing the effectiveness of the causal pathway.

Fig. 4
figure 4

Illustrative scenario for monitoring of the spread of a gene drive system from a release location. A In this example, each ring (black circle) is a 5 km radial distance from the release location (black circle at centre). The area between rings is defined as an annulus, identified here by sequential numbering from the release location. The annulus surrounding the release location is the release zone (light-blue ring surrounding black circle of release location). For the purposes of this illustration, it is assumed that modelling indicates that the rate of spread of the gene drive system is 20 km per annum from the release zone. The duration of this initial field trial would be 2 years. In this particular trial design scenario, entomological and epidemiological measurements could be compared between the release location and control locations (four navy blue circles) in ninth annulus (medium-blue). A buffer zone of the second to eighth annuli (grey) minimizes the potential for ‘spillover’ of the gene drive system between the release and control locations. For the purposes of measuring spread of the gene drive system, either larvae or adult mosquitoes could be tested for the presence of the transgene at monitoring locations every 5 km along transects running northwest to southeast and northeast to southwest from the release location (yellow stars), as well as in buffer zone habitations (purple circles). B In an example of a potential adaptive trial design element for monitoring, the gene drive system is detected in mosquitoes at monitoring locations the first, second, third, and fourth annuli (light blue stars) but also at a buffer zone habitation in the seventh annulus (light blue circle) C Additional adaptive monitoring locations (red stars) are introduced at the eighth and ninth annuli between the two transects to ensure as accurate as possible monitoring of both the rate and variability of spread of the gene drive system

In trial design, “blinding” is the concealment of the allocation of clusters to experimental or control arms from investigators and study participants [18]. This is desirable whenever possible to minimize the introduction of bias into site selection, data acquisition or reporting, or from differences human behaviour at control and experimental locations. Where full blinding is not feasible, it may still be possible to conduct portions of the trial, for example laboratory analyses, under blinded conditions [18].

Adaptive trial design

The attributes of indefinite spread and persistence of low-threshold gene drive prompt consideration of alternative types of more flexible trial design, or “adaptive trial design”, than those used for more conventional vector control interventions [93,94,95,96,97] (Fig. 5). In the context of clinical trials of pharmaceuticals, biologics, and health system interventions, adaptive trial design first gene drive field trial protocol incorporates the possibility of incremental escalation to more elaborate or complex goals once initial primary goals had been successfully achieved.

Fig. 5
figure 5

Illustrative example of a potential adaptive trial design for initial field trials of low threshold gene drive. Considering the intended potentially indefinite persistence and spread of the gene drive system in target populations, the first field trials could be divided into two Phases, building on the WHO guidance framework for the evaluation of GMMs [12]. Phase 2A would initially focus on the primary goals of assessing the increase in frequency of the gene drive system at, and the rate of its spread from, the release location but would also involve initiation of baseline data collections for subsequent assessment of Phase 2B entomological (‘@3’) and Phase 3 epidemiological (‘@4’) endpoints. Phase 2A of the trial could involve release of the gene drive system in a single vector species or multiple ones. Should the primary goals of Phase 2A be achieved, Phases 2B and 3 could be activated to allow the parallel assessment of entomological endpoints and epidemiological endpoints, potentially with expanded releases of the gene drive system in a single vector species or multiple ones Additional adaptive trial design elements could include flexibility in the numbers of mosquitoes and frequencies of release to achieve self-sustaining transmission of the gene drive system (‘@1’), adapting monitoring (‘@2’; see also Fig. 4); and potential to extend the duration of the trial where efficacy and safety assessments support this (‘@5’)

On one level, adaptive design for gene drive research programmes could involve initial phases that separately allowed assessment of modular components of a gene drive system that would precede the assessment of a fully integrated low threshold gene drive system [12]. For example, the components of a gene drive system could be tested without the need, or even the possibility, for their propagation beyond the immediate trial area by using modular systems where the gene drive and anti-pathogen effector gene functions are separate genetic components [98,99,100,101]. If earlier phases involving non gene drive components were successful, such a genetically confined approach would then proceed to later phases featuring self-sustaining gene drive (see Table 1).

On another level, developers could seek provisional and conditional regulatory approval and stakeholder support for a comprehensive protocol beginning with small-scale releases to test genetic efficacy in Phase 2A, then progressing to an expanded set of releases to assess entomological (Phase 2B) and epidemiological efficacy (Phase 3) subject to satisfactory genetic efficacy outcomes [12] (Fig. 5). Nevertheless, if additional locations were to be enlisted as the trial progresses, then their assignment as release or control clusters would need to be random; alternatively, groups of additional locations could be randomized to release or control clusters and treated as a stratum [18]. Importantly, if additional locations were assigned on the basis on interim analysis of data, the imposition of more stringent p-values on primary endpoints might be needed [18].

In such a scenario, formal permission for the first releases in Phase 2A would initiate an assessment of the increase in frequency of the gene drive system in target populations and its spread from the release locations, with the simultaneous activation of baseline entomological measurements and recruitment of human participants for a baseline assessment of epidemiological efficacy at Phases 2B and 3 of the trial (Fig. 5 ‘@3’ and ‘@4’). Successful outcomes from the Phase 2A of the trial would then activate Phases 2a and 3 of the trial involving additional field releases and parallel assessments of entomological and epidemiological endpoints. An alternative scenario could involve the sequential, rather than parallel, assessment of entomological and epidemiological efficacy in the trial, whereby successful entomological outcomes in Phase 2B would then activate the assessment of epidemiological endpoints in Phase 3.

In either of the above two scenarios for adaptive field release trials, the initial release locations could only form part of a cRCT if they are part of the random allocation of sites to either intervention or control. One way that this could be achieved would be to begin the field trial with a small, even number of locations and group these in pairs, so that the two members of each pair were as similar as possible to each other and then randomly allocate locations within each pair to release or control arms of the trial. For example, initially the gene drive field trial could commence with only one pair of locations, with the initial releases taking pace in the randomly selected release location. Additional pairs of study locations could be added as the size and complexity of the trial expanded, but always with random allocation of locations to release and control arms of the trial, until there was a sufficient build-up of clusters to a fully powered cRCT. In this way, entomological and epidemiological comparative evaluations of the two trial arms could commence once outcomes from earlier genetic efficacy evaluations were incorporated into their optimal design and implementation. By monitoring the spread of the gene drive system from the release to control locations from the beginning of the trial at the earliest randomly allocated pairs, the appropriate size of the buffer zone between locations within pairs could be determined, so that locations with pairs that would be subsequently added to the trial could be sufficiently separate from one another to avoid spillover.

Assessing genetic efficacy in Phase 2A

Given the pre-release absence of the gene drive system from wild populations, an obvious ambition in initial field trials will be to investigate genetic efficacy endpoints. Observation of an increase in the frequency of the gene drive system in target populations after cessation of releases would provide one aspect of the successful demonstration of genetic efficacy. This could be based initially on the measurement of the frequency of the gene drive system in mosquitoes at the release locations and proximal environs over an extended period (Fig. 2 and Box 3). It would be useful to understand the basis for any differences between the predicted increase in frequency of the gene drive system and those observed empirically in the field.

Assessing entomological efficacy in Phase 2B

The WHO recommends the gathering and assessment of baseline entomological data from study locations that are relevant to efficacy, such as vector species composition and rates of malaria transmission, which can incorporate variation in endpoints over multiple seasons and transmission cycles [12, 68]. It also proposes that questions identified in ERA should inform the types of baseline data that will be required to characterize the impact of the field releases on health and the environment. Furthermore, it points out that mathematical modelling and network analyses also could identify significant species interactions that will inform baseline data gathering. Considerations could include: (i) key ecological data, (ii) mosquito and parasite genetic variation, (iii) seasonal variation in vector species densities, (iv) fitness parameters such as fertility, fecundity, and survival, (v) locations of aquatic habitats, (vi) locations of swarming sites, (vii) levels of migration of the target wild type mosquitoes, and (viii) mosquito biting behavior [12]. In addition, potential confounding factors that could alter mosquito densities or malaria transmission at the trial locations also should be considered as part of baseline measurements, including (ix) human impacts on the landscape such as habitation, irrigation, and agriculture, (x) rainfall, temperature, climate, and geography, and (xi) existing or planned vector control measures (Fig. 2). These confounding factors can be addressed by appropriate randomization of locations to control and release arms of the field trial.

In contrast to genetic efficacy, entomological efficacy would be assessed using populations as the unit of analysis and would typically occur once a gene drive system has reached, or is close to reaching, fixation in target populations (Figs. 1 and 2; Box 3). To maximize the potential for the first gene drive field release trials to be as informative as possible, specific research questions and endpoints should be chosen for which anticipated effect sizes are as large, and variability as low, as possible (Table 1). Decisions on trial design also could be based on observational mechanistic linkages within the causal pathway, for example, correlation of increased frequency of the gene drive system with reduced entomological inoculation rate (EIR) in target vector populations. However, previous guidance from the Vector Control Advisory Group (VCAG) of the WHO recommends statistically powering entomological endpoints for trials [15].

For population suppression gene drive, comparison of the density of target populations of mosquitoes at release and control locations will be an important entomological parameter to assess [70, 71, 74, 75, 81, 90, 151]. For example, a battery of ovitraps and BG sentinel traps covering both control and release locations were used to ascertain measurements relating to adult mosquito density in a trial of GMMs to control Aedes aegypti in Brazil [114]. For population modification gene drive, comparison of sporozoite rate of females in target populations at release and control locations will be a key assessment, in addition to associated biting rates and densities in the same populations to provide an overall measurement of EIR [70, 71, 74, 81]. In theory, non-target vector species could serve as entomological controls in field studies; however, species composition can vary considerably across nearby locations and seasonally and the intervention itself could, for example, affect competitive interactions between vector species [23]. The fraction of total EIR that could be reduced as a result of gene drive in the target species could then be estimated from baseline data. Use of genetic information in mosquito target populations, such as measurement of nucleotide diversity or linkage disequilibrium, may also provide an additional and useful method of gauging the effectiveness of population suppression gene drive, as such genomic parameters depend on population size [152].

As there is likely to be significant variation in densities of target populations across study locations and over time due to variation in environmental factors such as rainfall as well as sampling biases imposed by collection methods [15, 109, 153,154,155], field implementation of a statistically powered assessment of entomological endpoints could also require considerable resources. Although entomological outcomes are not by default reliable predictors of epidemiological outcomes, such entomological measurements could contribute high-quality data that should lead to important correlations with any observed epidemiological impacts [17, 156, 157]. Accurate and representative entomological data collection from field sites that have been selected as potential release or control locations would be highly valuable in informing whether entomological outcomes from initial field trials are attributable to the gene drive system and distinct from stochastic background variation [12].

VCAG has suggested that six to ten randomly selected households or trap nights per site, with entomological measurements compared in a time series over the course of a season rather than as a simple before-and-after measurement [15]. Drawing on baseline field entomological data, statistical tools are under active development that can inform decision-making on choices of entomological endpoints, and to inform power calculations on releases in single versus multiple vector species for initial field trials of low-threshold gene drive. As with the design of any cRCT, the amount of variation in entomological measurements between trial locations also will be a factor in determining the number of clusters required in control and intervention arms of the field trials involving low threshold gene drive [15, 50, 158,159,160].

In addition to entomological endpoints measuring efficacy, any entomologically based risks identified in pre-release ERA of the gene drive system will most likely also need to be assessed at this phase of initial cRCTs. These could include measurements evaluating whether (i) there were any adverse effects from detection of the gene drive system in other sibling species sympatric to the target mosquito populations, (ii) there were any effects on non-target organisms that adversely impacted health or ecosystem services, (iii) in the case of release of a population suppression gene drive, there were any effects on competitor or predator species of target mosquito populations that adversely impacted health or ecosystem services, (iv) in the case of population modification, there were any changes in pathogenicity or transmissibility of Plasmodium falciparum or in other species of malaria pathogens, such as Plasmodium vivax, Plasmodium ovale, or Plasmodium malariae that adversely impacted health or ecosystem services, or (v) there was any evidence of gene flow into non-reproductively compatible species of insect and that caused adverse impacts (Table 1).

Assessing epidemiological efficacy in Phase 3

As with entomological efficacy, epidemiological efficacy would be assessed using the population or cluster as the unit of analysis once a gene drive system has reached, or is close to reaching, fixation in target populations. While successful implementation of the initial field trials of low-threshold gene drive systems may be favored when objectives are simple and focused on addressing essential efficacy and safety questions, such as whether the system can increase in frequency and spread in target populations, initial trials should also provide sufficient information, experience, and confidence to optimize field releases for later, more complex, and larger field trials. Similar incrementally expanding approaches are used as standard practice in Phase I-IV clinical trials of new pharmaceuticals or biotechnology products [13, 161].

Definitive epidemiological assessments or results could be confounded by other vector control interventions being used in the study area. VCAG has recommended that trials should be statistically powered for epidemiological endpoints [15]. Epidemiological efficacy of the gene drive system may be more difficult to establish, but easier to implement, when relying solely on passive case detection of malaria prevalence [12, 15, 162], as this measures the rate of both pre-existing and new symptomatic infections in the population, and is prone to variability in the health-seeking behavior of people, the quality of diagnostic services available, and administrative practices, such as record-keeping and attributions of correct residential location to patients. Therefore, while it remains possible that information on cases of malaria at release and control locations of the first gene drive field trials could be captured by passive case detection at health centers or in routine surveillance programmes, the prevalence of infection would be more robustly assessed by means of a representative randomly selected cross-sectional survey, usually a household survey, in all clusters, by measuring infection status of individuals [12, 70, 75, 76, 162] (Fig. 2).

Such data would be relevant for both population suppression and population modification approaches. In addition, active case detection is more likely to have the power to detect significant changes in the incidence of infection and disease [12, 81, 90, 163]. However, this approach might be considered as a more expensive and time-consuming investment in initial field trials as it would most likely require the recruitment and treatment of participants with anti-malarial medications to eliminate any pre-existing cases of parasitaemia ahead of the commencement of the gene drive releases and assessment. Nonetheless, such recruitment is routinely the case in other cRCTs for malaria vector control. Therefore, given that the fundamental objective in the development of low-threshold gene drive in Anopheles mosquitoes is to reduce malaria transmission, active case detection of malaria incidence would likely produce robust and accurate assessments of epidemiological efficacy [12].

Additional aspects of morbidity associated with malaria, such as anaemia and splenomegaly, could also be assessed in trial participants at control and release locations [70, 75] (Fig. 2), although in most published cRCTs these endpoints typically seem insufficiently sensitive to changes in malaria transmission. The relationship between parasite prevalence and clinical incidence of malaria appears to be more complex than a simple linear one, so that simultaneous measurements of both these endpoints might be contemplated in ideally designed vector control trials [50, 164].

In addition to epidemiological endpoints measuring efficacy, any epidemiologically based risks identified in pre-release ERA of the gene drive system will most likely need to be assessed in this stage of initial cRCTs. These could include measurements evaluating whether there are changes in (i) the incidence or prevalence of malaria before and after field releases or at release versus control locations, (ii) the incidence or prevalence of other diseases before and after field releases or at release versus control locations, (iii) human behavioural responses towards conventional vector control measures such as bednets as a result of releases of the gene drive system, or (iv) perspectives of the local or wider communities to the specific intervention and wider gene drive technology (Table 1).

While the time to impact of releases of gene drive systems for malaria vector has been investigated in a number of modelling studies, most of these have focused on post-trial implementation that have typically involved annual releases of low numbers of mosquitoes at relatively dispersed locations yielding time to entomological or epidemiological impacts on the order of one to two years after releases have commenced [23, 99, 165]. However, a recent modelling study of initial field releases of a population modification gene drive system on an island setting suggested that, under at least some circumstances, a significant reduction in malaria incidence could be anticipated within three to six months [148]. The development of further modelling tools and studies exploring a range of different release rates, frequencies, and spatial structures should inform expectations on how rapidly efficacy might be observed in initial gene drive field trials. Another important consideration may be whether one particular kind of gene drive may be more effective than another in specific malaria transmission settings. This in turn may affect the outcomes to be measured. Indeed, the epidemiological impact may depend on the vector species being targeted by the gene drive system and the species composition in the study area (Box 2).

It also could be possible in adaptive field trial design to extend the duration of the trial depending on emerging data (Fig. 5 ‘@5’). For example, in the case of population modification gene drive, the gene drive system could continue to spread from the release location without its effector gene [166]. For population suppression gene drive, mosquito populations may be reduced but not eliminated, allowing the gene drive to continue to spread through target populations [165].

Such examples underscore the importance of the outputs of ERA of the release of the gene drive system in playing a key role to allow regulatory and stakeholder decision-making around the acceptability of the potential risks versus the potential benefits from a low-threshold gene drive system, including when and if the trial should be considered to have ended. Nonetheless, any flexibilities in the duration of the trial would have to be subject to clearly defined stopping rules, which could impose penalties of lower p-values for statistical significance any time an interim analysis were to be carried out [18]. In addition, some form and level of post-trial field monitoring of the gene drive system, at the release locations and potentially distally, may be required by decision-makers [12].

Considering the indefinite persistence and spread of low-threshold gene drive in the field, whether initial field trial focus exclusively on measurement of genetic efficacy, or on broader aspects of the causal pathway, how and when epidemiological efficacy will eventually be assessed will be an essential consideration before decisions on any field trial protocols are finalized and implemented.

Conclusions

A fundamental question for field trials of low-threshold gene drive for malaria vector control will be whether the intended causal pathway leading from release of gene drive mosquitoes to reduction in malaria transmission will be effective. Key decisions for initial trials will focus on whether primary objectives should focus exclusively on assessments of genetic, entomological, or epidemiological efficacy, or combinations thereof. While effective conduct of initial field trials of low-threshold gene drive systems may be favoured when objectives are simple, such as whether the system can increase in frequency and spread in target populations, initial trials also should be designed and implemented to provide sufficient breadth and depth of information, experience, and confidence to optimize field releases for later, more complex, and larger field trials involving more robust epidemiological assessments. Because spread and persistence of low-threshold gene drive systems in target populations are intended features, adaptive trial design offers a potentially constructive and flexible approach to incremental testing of the causal pathway. Such an approach could facilitate the first field trials of low threshold gene drive to be staged incrementally so that genetic efficacy would first be assessed and, if proven, activate further stages of testing involving entomological or epidemiological endpoints, or both. How and when epidemiological efficacy will eventually be assessed will be an essential consideration before decisions on any field trial protocols are finalized and implemented, regardless of whether initial field trials focus exclusively on measurement of genetic efficacy, or on broader aspects of the causal pathway. Statistical and modelling tools that will support decision-making on choices of such trial locations and endpoints, adaptive trial design, robust power calculations, effective monitoring tools and methodology, and field operational responsiveness to emerging monitoring data during trials, are currently under active development. The considerations here advance the realization of developer ambitions for the first field trials of low-threshold gene drive for malaria vector control within the next 5 years.

Availability of data and materials

Not applicable.

Abbreviations

ACT:

Artemisinin-based combination therapy

AUDA-NEPAD: :

African Union Development Agency-New Partnership for Africa's Development

BG:

Biogents™ (sentinel traps)

CDC:

U.S. Centers for Disease Control and Prevention

cRCT:

Cluster randomized controlled trial

DSBM:

Data and Safety Monitoring Board

eDNA:

Environmental DNA

EIR:

Entomological inoculation rate

ELISA:

Enzyme-linked immunosorbent assay

ERA:

Environmental risk assessment

FNIH:

Foundation for the National Institutes of Health

GCP:

Good Clinical Practice

GMM:

Genetically modified mosquito

HLC:

Human landing catch

ICH:

International Council for Harmonization

IRS:

Indoor residual insecticide spraying

ITN:

Insecticide-treated bed net

LAMP:

Loop-mediated isothermal amplification

LLIN:

Long-lasting insecticidal bed net

MRR:

Mark-release-recapture field study

PCR:

Polymerase chain reaction

PSC:

Pyrethrum spray catch

UAV:

Unmanned Aerial Vehicle (“drone”)

VCAG:

Vector Control Advisory Group of the WHO

WAIVM:

West African Integrated Vector Management Programme

WAHO:

West African Health Organization

WHO:

World Health Organization

WMA:

World Medical Association

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Acknowledgements

We thank Abdoul-Azize Milogo, Ace North, Adrian Leach, Alexandre Quach, Alima Qureshi, Ana Kormos, Andrea Crisanti, Brian Tarimo, Camilla Beech, Charles Guissou, Emma Maynard, Franck Yao, Fred Tripet, Greg Lanzaro, Immo Kleinschmidt, John Marshall, John Mumford (R.I.P.), Katie Willis, Laura Norris, Lea Pare Toe, Lodney Nazare, Mamadou Coulibaly (R.I.P.), Nick Jewell, Nicole Achee, Nigel Beebe, Sam O’Loughlin, Scott O’Neill, Tin-Yu Hui, Tom Churcher, and Tony Nolan for their contributions to this work.

Funding

JBC, AB, AD, PAH, JKK, ARMcK: supported by grant from the Bill & Melinda Gates Foundation and Open Philanthropy. GC, NW: supported by the Bill and Melinda Gates Foundation grant OPP1158151. AAJ: supported by The University of California Irvine Malaria Initiative (UCIMI), the Bill and Melinda Gates Foundation (INV-043645), National Institutes of Health (AI170692) and an anonymous donor. AAJ is a Donald Bren Professor at the University of California, Irvine. MRS and FR: supported by grant INV-008525v from the Bill & Melinda Gates Foundation.

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MRS and FR conceptualized the study. JBC, AB, GC, AD, TH, PAH, AAJ, JKK, DWL, AM, ARMcK, MS, NW, and FR contributed content and perspectives to deliberations and the manuscript. JBC and FR drafted and revised the manuscript. JBC, AB, GC, AD, TH, PAH, AAJ, JKK, DWL, AM, ARMcK, MRS, NW, FR reviewed and approved the final manuscript.

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Correspondence to John B. Connolly.

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JBC, AB, AD, PAH, JKK, ARMcK: members of Target Malaria not-for-profit research consortium; awardees of travel grants from FNIH.

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Connolly, J.B., Burt, A., Christophides, G. et al. Considerations for first field trials of low-threshold gene drive for malaria vector control. Malar J 23, 156 (2024). https://0-doi-org.brum.beds.ac.uk/10.1186/s12936-024-04952-9

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