**2. Population modification for malaria control**

#### **2.1 What is population modification?**

Population modification is the concept of incorporating genes or genetic elements in vector species that increase their refractoriness to the pathogens they transmit thereby inhibiting transfer of the pathogens to host species (**Figure 3**). Population modification was first described in the contemporary literature using the term 'population replacement' by Christopher Curtis in 1968 [26]. Due to misinterpretations of population replacement and negative connotations of the term 'modification' related to cultural perspectives on genetically-modified organisms (GMOs), a third term, 'population alteration', also was proposed [27]. The early conceptions of population modification were made prior to the discovery and refinement of current gene-drive technologies, however, the original concept as proposed by Curtis suggested the need for a mechanism to elicit fixation of the favorable genes in a population. The advancements and development of genetic-engineering techniques to inhibit *Plasmodium* spp. have occurred in

#### **Figure 3.**

*Outcomes anticipated from genetic control approaches. Vector control strategy utilizes genetic-engineering technology with gene drive via two different approaches, population modification/alteration (top) or population suppression (bottom). In both approaches, the transgenic mosquitoes qualified for releases should carry at least three components: the gene drive system, the marker and the effector or suppression component aiming at reducing the vector competence or the vector population, respectively. The anticipated outcome for the population modification/ alteration approach is that the treated population become refractory to pathogen as the effector genes spread into the population; whereas with the population suppression approach the anticipated outcome would be the reduction or elimination of whole population. In both cases, the goal is to break the parasite cycle in the mosquito stages.*

parallel with the development of gene-drive technologies and today proof-of-principle concepts for population modification strains exist in both the African malaria vector, *An. gambiae* and the Indo-Pakistani vector, *Anopheles stephensi* (**Table 1**) [28–31].


*Cp, carboxypeptidase gene promoter; Vg, vitellogenin gene promoter; 1C3, 2A10: single-chain antibodies; WT: wild-type.*

#### **Table 1.**

*Proof-of-principle gene-drive systems with and without antimalarial effectors in Anopheles mosquitoes for population modification/alteration strategy.*

### **2.2 Population modification vs. population suppression**

Population suppression is an alternative strategy to population modification that utilizes genetic-engineering technologies to reduce vector number and therefore reduce pathogen transmission (**Figure 3**). This can be achieved by diminishing the fitness or distorting sex ratios so that the vector populations reduce in number and eventually go extinct locally. Similar to population modification, proof-of-principle concepts also exist for population suppression in *An. gambiae* [32–34]. The advantages and disadvantages of both population modification and suppression drives are described succinctly in a recent review [35]. One advantage of suppression drives is that they create a rapid reduction in vectorial capacity by immediately having suppressive effects on the targeted mosquito population (reductions in entomological inoculation rate and human biting rate) and thus quickly reducing the basic reproductive rate (R0) of malaria. Another advantage is that suppression drives also will reduce transmission of all possible pathogens vectored by the target species. Unanswered questions that give cause for concern are what happens to the empty ecological niche left by the vector species, and will suppression to extremely low population levels allow re-introduction of wildtype mosquitoes that then transmit

the pathogen to a more highly susceptible human population? In contrast, population modification strategies are not likely to have as much of an immediate effect on the vectoral capacity and subsequent R0 as the drive system takes time to introduce the effectors into the population and an R0 < 1 is likely to require a sufficient proportion of the population to carry effector genes [36]. However, population modification strategies do not leave an empty ecological niche and the introduced anti-parasite genes are anticipated to remain stable in a population and this could mitigate the role that re-introduction of infectious wild-type mosquitoes might have in the local population. Population modification is predicted to be sustainable during the control, pre-elimination, elimination, and prevention-of-reintroduction stages of local malaria elimination and thereby provide a cost-effective method for maintaining local elimination [37]. It is expected to be useful in the elimination phase by complementing other strategies that reduce mosquito population sizes. Some potential disadvantages include the potential to select parasites resistant to a single effector mechanism. One strategy to mitigate development of parasite resistance to effectors is by including multiple effector components that target various stages of the parasite development cycle within the mosquito [38]. The effector components used may be exogenous, such as single-chain antibodies (scFvs), endogenous, such as a manipulation of genes associated with mosquito innate immunity or a combination of both [38–41]. A second strategy to mitigate parasite resistance to population modification strains is to reduce the parasite population prior to and during the field release of the modified mosquitoes so that there is less opportunity for resistance to develop due to lower replication rates in the parasite population [42]. Encouragingly, both strategies to mitigate parasite resistance can be combined to provide pathogens with a more insurmountable barrier to developing resistance.

Population modification and population suppression vary in their strengths and weaknesses so a complementary approach that involves the sequential application of both technologies can be proposed (**Figure 4**). This strategy maximizes the benefits of both approaches and lowers their respective hurdles to long-lasting success. The complementary approach includes an initial field release of a population suppression strain that will act to quickly reduce the local population of vectors and their associated population of parasites. When the population structure of native vectors has been sufficiently disrupted by the suppression strategy, the low level of individuals becomes more susceptible to events that may inhibit its ability to persist long term. For example, a re-introduction of wild-type individuals can occur, and these may overwhelm any low levels of remaining drive individuals, or individuals with driveresistant alleles may build up over time inhibiting future suppression [43]. At this point, when a suppression system has driven the population to levels near extinction, a modification line can be introduced for maximal effect. Allowing a population replacement mosquito line to form the new population of mosquitoes prevents any negative ecological effects that may have occurred due to an empty ecological niche. It also allows the population modification drives to become established in an environment with a minimized risk for resistance to the transgene introduction. The effector genes will be less prone to having pathogen-based resistance develop as the natural pathogen population will have been greatly diminished by the suppression system, and lower pathogen reproduction numbers lower the likelihood of randomly-generated resistance conferring mutations in the pathogens. In the absence of threats from resistance, the only further threat faced by the population modification strain is long-term stability of the effector elements. However, new effector elements can be developed

#### **Figure 4.**

*Vector control with population modification and population suppression complementary approach. Proposed strategy combining sequential releases of mosquitoes with population modification and population suppression drives. The combined approach initiated with releasing population suppression gene-drive mosquitoes, which theoretically reduce the whole mosquito population in the treated area. Follow up with releasing of population modification gene-drive mosquitoes, this strategy ensures avoidance of an empty niche or re-introducing of wild mosquitoes that are susceptible to the malaria parasite. Black: wild-type mosquito; yellow: transgenics mosquitoes with suppression drive; Green: Transgenic mosquitoes with population modification drive.*

carefully as the needed window for protection resulting from the complementary approach is likely to be much longer than either approach alone.

#### **3. Engineering refractory mosquitoes**

The malaria parasites go through a multi-staged life cycle within their mosquito vectors (**Figure 5**). After the female *Anopheles* mosquito bites an infected human, if ~1000 *Plasmodium* male and female gametocytes are ingested with the blood meal, subsequent fertilization produces as many as 25 diploid zygotes. The zygotes mature to a motile form, the ookinete, that penetrates the mosquito midgut epithelium where only a few (<5) will mature to oocysts. Mitotic and meiotic divisions occur in the oocysts to give rise to several hundred to thousands of haploid sporozoites. The sporozoites (~5000) are released into the hemolymph (the mosquito open circulatory system) within 10–14 days post infection. The sporozoites then travel through the hemolymph to reach and invade the salivary glands and are transmitted as the infectious form of the parasite to a new human host during subsequent bites.

A synthetic approach was used in our laboratory to develop the anti-parasite effector genes and introduce these desired traits into the target genomes to generate the genetically-engineered mosquitoes (GEMs) [37]. This approach has several advantages, for example, the components of a synthetic construct can be relatively small, their functions are more fully known and the site in the mosquito genome where they will be located can be characterized or determined prior to genome integration. A synthetic cassette for population modification has two main components: (1) promoters and (2) antimalarial effector genes.

#### **Figure 5.**

*Malaria developmental pathway and compartments for blocking parasite development. Gametes are ingested with the blood meal. They differentiate, fertile and form a zygote. The zygote develops into a motile form, the ookinete, that then invades the mosquito midgut epithelium. There it develops into an oocyst in which many sporozoites are generated. These burst into the hemolymph and migrate to the salivary glands. From there the sporozoites can be transmitted to a new host during the next blood meal. The midgut compartment allows access to the gametes, zygotes, ookinetes and oocysts. The hemolymph and salivary gland compartments allow access to the sporozoites (image adapted from Isaacs et al. [38]).*

#### **3.1 Promoters**

Promoters are regulatory DNA sequences that will drive the expression of a transgene (a marker or an antimalarial effector) in mosquitoes. During its development in the mosquito, the malaria parasite occupies three main compartments: midgut lumen, hemocoel and salivary gland lumen (**Figure 5**). Expression of the anti-parasite genes in these compartments is crucial to block their transmission and several tissue-specific promoters have been identified and used in mosquito transgenesis. These include control sequences for a gene encoding a carboxypeptidase, a digestive enzyme, and AgAper1, a peritrophic matrix protein, which are activated in response to a blood meal [44–46]. The vitellogenin-encoding gene promoters drive strong expression in the fat body and hemocoel [47, 48]. A hemocyte-specific hemolectin (hml) gene promoter and three salivary gland-specific promoters, (*Apyrase* [*Apy*], *maltase-like I* [*Mal1*] and *anopheline antiplatelet protein* [*AAPP*] promoter), also have been developed [49–52]. Ubiquitously-expressed gene promoters (*heat-shock protein 70* [*hsp70*], *actin 5C*, and *ubiquitin* and *polyubiquitin*) also could be used to drive expression of the effector genes, however, their generalized expression may impose a higher fitness load in the GEM [53–55]. These gene promoters have been used effectively to drive the expression of genes encoding generally benign fluorescent proteins as dominant markers for transgene presence.

#### **3.2 Antimalarial effector genes**

The effector molecules can be classified into four groups depending on their mode of action.

i.Parasite blocking: exogenous molecules that eliminate the parasites such as antimicrobial peptides from the immune system of other insects (gambicin, defensin, cecropin) or other arthropods (scorpine). Natural and synthetic lytic peptides such as angiotensin II, magainins, Shiva-1, Shiva-3 and gomesin have been used to generate refractory *Plasmodium* mosquitoes [56–62].


The identification and characterization of efficient anti-*Plasmodium* effector genes is essential to generate refractory mosquito phenotypes. Expression of these genes may result in GEMs being less competitive than their wild-type counterparts. Ideally, the effector molecules should interfere with parasite transmission without imposing a fitness cost to the mosquito. Furthermore, these genes clearly impose selection pressures on the parasites and the emergence of parasites resistant to the effector molecules could occur. As discussed previously, this may be mitigated by using combinations of multiple anti-*Plasmodium* effector proteins with different modes of action that can overcome the possibility of parasite resistance. Recently, Dong et al. (2020) showed that it is possible to generate a transgenic line (MultiEff) expressing simultaneously five anti-*Plasmodium* effectors (melittin, TP10, shiva1, EPIP, and scorpine) with a significant parasite-blocking effect at the pre-oocyst stage and low fitness cost [68].
