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Field Trials of Gene Drive Mosquitoes: Lessons from Releases of Genetically Sterile Males and Wolbachia-infected Mosquitoes


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The discovery of CRISPR-based gene editing and its application to homing-based gene drive has been greeted with excitement, for its potential to control mosquito-borne diseases on a wide scale, and concern, for the invasiveness and potential irreversibility of a release. At the same time, CRISPR-based gene editing has enabled a range of self-limiting gene drive systems to be engineered with much greater ease, including (1) threshold-dependent systems, which tend to spread only when introduced above a certain threshold population frequency, and (2) temporally self-limiting systems, which display transient drive activity before being eliminated by virtue of a fitness cost. As these CRISPR-based gene drive systems are yet to be field-tested, plenty of open questions remain to be addressed, and insights can be gained from precedents set by field trials of other novel genetics-based and biological control systems, such as trials of Wolbachia-transfected mosquitoes, intended for either population replacement or suppression, and trials of genetically sterile male mosquitoes, either using the RIDL system (release of insects carrying a dominant lethal gene) or irradiation. We discuss lessons learned from these field trials and implications for a phased exploration of gene drive technology, including homing-based gene drive, chromosomal translocations, and split gene drive as a system potentially suitable for an intermediate release.
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Chapter 2
Field Trials of Gene Drive Mosquitoes:
Lessons from Releases of Genetically Sterile
Males and Wolbachia-infected Mosquitoes
John M. Marshall and Váleri N. Vásquez
Abstract The discovery of CRISPR-based gene editing and its application to
homing-based gene drive has been greeted with excitement, for its potential to
control mosquito-borne diseases on a wide scale, and concern, for the invasiveness
and potential irreversibility of a release. At the same time, CRISPR-based gene
editing has enabled a range of self-limiting gene drive systems to be engineered with
much greater ease, including (1) threshold-dependent systems, which tend to spread
only when introduced above a certain threshold population frequency, and (2) tem-
porally self-limiting systems, which display transient drive activity before being
eliminated by virtue of a tness cost. As these CRISPR-based gene drive systems are
yet to be eld-tested, plenty of open questions remain to be addressed, and insights
can be gained from precedents set by eld trials of other novel genetics-based and
biological control systems, such as trials of Wolbachia-transfected mosquitoes,
intended for either population replacement or suppression, and trials of genetically
sterile male mosquitoes, either using the RIDL system (release of insects carrying a
dominant lethal gene) or irradiation. We discuss lessons learned from these eld
trials and implications for a phased exploration of gene drive technology, including
homing-based gene drive, chromosomal translocations, and split gene drive as a
system potentially suitable for an intermediate release.
Keywords Trial · Gene drive · Mosquitoes · Genetically sterile males · Wolbachia
J. M. Marshall (*)
Divisions of Epidemiology and Biostatistics, School of Public Health, University of California,
Berkeley, CA, USA
Innovative Genomics Institute, University of California, Berkeley, CA, USA
V. N. Vásquez
Energy and Resources Group, University of California, Berkeley, CA, USA
Berkeley Institute for Data Science, University of California, Berkeley, CA, USA
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
B. K. Tyagi (ed.), Genetically Modied and other Innovative Vector Control
2.1 Introduction
The discovery of CRISPR and its application as a gene editing tool has enabled gene
drive systems to be engineered with much greater ease (Doudna and Charpentier
2014; Champer et al. 2016). Recent attention has focused on homing-based drive
systems and their potential to control mosquito-borne diseases on a wide scale, either
by spreading disease-refractory genes (Gantz et al. 2015) or by spreading genes that
confer a tness load or sex bias thereby suppressing mosquito populations
(Hammond et al. 2016; Kyrou et al. 2018). However, there is growing awareness
of the invasiveness of homing-based drive systems (Noble et al. 2018) and interest in
alternatives that could be conned to partially isolated populations and remediated
properties that are well aligned to the conduct of eld trials (Marshall and Akbari
In addition to homing-based gene drive, the increased ease of gene editing has
advanced the entire eld of gene drive, including systems that, by design, limit their
spread in space and time (Marshall and Akbari 2018). Such systems would ideally be
capable of enacting local population control by: (a) effectively spreading into
populations to the extent required to achieve the desired epidemiological or ecolog-
ical effect and (b) being recallable from the environment in the event of unwanted
consequences, public disfavor, or the end of a trial period. Two varieties of these
systems have been recently engineered: (1) threshold-dependent systems that tend to
spread when introduced above a certain population frequency (Akbari et al. 2013;
Buchman et al. 2018) and (2) temporally self-limiting systems that display transient
drive activity before being eliminated by virtue of a tness cost (Gould et al. 2008;Li
et al. 2020).
In this chapter, we discuss considerations for eld trials of gene drive systems,
with a specic focus on connement and reversibility criteria, and lessons learned
from other genetics-based and biological control systems (Table 2.1). We pay
special attention to reciprocal chromosomal translocations (Buchman et al. 2018),
as an example of a threshold-dependent system that is conneable and reversible,
and then extend our consideration to CRISPR-based homing gene drive systems and
temporally self-limiting systems, such as split gene drive (Li et al. 2020), which
could be used as conneable and reversible intermediate systems in a development
pathway of homing-based systems. While these gene drive systems are yet to be
trialed in the wild, lessons can be learned from trials of several varieties of sterile
male mosquitoes, specically, those sterilized through radiation (sterile insect tech-
nique, SIT), transfection with Wolbachia (incompatible insect technique, IIT)
(Zheng et al. 2019; Crawford et al. 2020), and release of insects carrying a dominant
lethal (RIDL) gene (Harris et al. 2011; Carvalho et al. 2015), as well as releases of
Wolbachia-infected mosquitoes for population replacement (Hoffmann et al. 2011).
We begin by discussing trials of these systems and discuss threshold-dependent,
self-limiting, and nonlocalized gene drive systems in this context.
22 J. M. Marshall and V. N. Vásquez
2.2 Lessons from Releases of Genetically Sterile Male
Releases of irradiated sterile male insects as a means of population suppression have
been discussed since the early twentieth century (Klassen and Curtis 2005), and a
transgenic version of this technology was the rst transgenic mosquito product to be
trialed in the eld (Harris et al. 2011). As the rst transgenic mosquito release, this
intervention has come under high levels of scrutiny and serves as an important case
study for potential releases of gene drive mosquitoes. The traditional SIT approach
involves mass-rearing insects and applying a carefully calibrated amount of radiation
such that their genetic material is mutated to render them sterile while still being able
Table 2.1 Genetics-based and biological mosquito control strategies and their potential to be
conneable and reversible
Strategy Variant Mechanism of action Conneable Reversible
Sterile insect
technique (SIT)
Ionizing radiation
Offspring of released
females and males are
Yes Yes
Wolbachia Incompatible
insect technique
Offspring of released
males are unviable
Yes, if no
Yes, if no
Spreads through popula-
tion due to cytoplasmic
Yes, for
to-high t-
ness costs
for high t-
ness costs
Release of
insects carrying
a dominant
lethal (RIDL)
Bisex RIDL
Both female and male off-
spring having the RIDL
allele are unviable
Yes Yes
Only female offspring
having the RIDL allele are
Yes Yes
CRISPR or other
Translocation heterozy-
gotes with unbalanced
chromosome sets are
unviable, leading to
bistable dynamics
Yes Yes
gene drive
drive systems
Bias inheritance by cleav-
ing a target sequence and
serving as a template for
DNA repair, effectively
turning a heterozygote into
a homozygote
but with
but with
Split gene drive
Components of drive sys-
tem are split across two
loci, leading to transient
drive when they co-occur
before being eliminated
due to a tness cost
Yes Yes
2 Field Trials of Gene Drive Mosquitoes: Lessons from Releases of Genetically... 23
to compete for female mates in the eld (Knipling 1955). Upon release, sterile males
(preferably the majority of released insects) seek out wild females, essentially
wasting their reproductive potential as the females produce no or signicantly less
viable offspring. Consecutive releases over a sufciently wide area result in less
productive matings and a progressive reduction in insect population size over
subsequent generations (Hendrichs and Robinson 2009).
The most widely celebrated application of SIT involved the use of ionizing
radiation to eradicate the screwworm y, Cochliomyia hominivorax, from North
Americaa program that began in 1957 following successful eld trials on the
Island of Curacaoand continues to this day to prevent reinvasion of the continent
(Wyss 2000; Klassen and Curtis 2005). In this intervention, large-scale releases of
sterilized insects led the screwworm y population in the USA to crash within a
decade. Subsequent releases progressively shifted the eradication zone southward,
eventually covering all of North and Central America by 2001 (Robinson 2002).
The success of the screwworm SIT project motivated the application of SIT to a
range of other insect pest species, including mosquito vectors of disease (Knipling
1968). Both irradiation and chemosterilization were initially explored for applica-
tions to mosquitoes, and in the 1960s and 1970s, large SIT eld trials were
conducted using chemosterilized Culex quinquefasciatus in India and Anopheles
albimanus in El Salvador (Klassen and Curtis 2005). The trial in India was halted in
the mid-1970s, following false accusations that the project was being used to collect
data to engage in biological warfare (Nature 1975), highlighting the importance of
effective community and political engagement for international biocontrol programs.
Nevertheless, benets of chemosterilization were demonstrated for this particular
intervention due to reduced tness costs as compared to irradiation.
A signicant advancement in SIT technology was ushered with specic DNA
changes introduced by the RIDL construct (Thomas et al. 2000; Alphey 2002).
Insects sterilized through mutagens are subject to a myriad of random genetic
mutations, which are invariably associated with signicant tness costs. In theory,
releases of insects carrying (in homozygous form) a dominant lethal gene (RIDL)
have essentially the same population impact as SITi.e., offspring of released males
are unviablealthough in a more controlled way that has potential for smaller
associated tness costs. Benedict and Robinson (2003) argued that a transgenic
version of SIT should be the rst application of transgenic mosquitoes in the wild
(as it was), both for enhanced efcacy and for biosafety featuresi.e., lethality-
inducing transgenes should be quickly eliminated from the environment, causing the
intervention to be reversible within a few generations. Quick elimination of
transgenes also leads to connement, since released mosquitoes can only travel so
far in a few generations.
Sterile insect approaches based on genetic engineering present more opportunities
than those based on mutagenesis, as genes and their associated traits can be modied
in a more precise way. The original RIDL strain in Aedes aegypti, OX513A, causes
lethality in both female and male offspring (bi-RIDL) (Thomas et al. 2000); how-
ever, an alternative construct was engineered soon after that only causes female
offspring to be inviable (female-specic RIDL, or fs-RIDL). This allows the
24 J. M. Marshall and V. N. Vásquez
population-suppressing trait to persist for a few more generations through the male
line, while continuing to suppress the female population, which is effective since
only female mosquitoes bite and transmit diseases to humans. Furthermore, the
introduced trait is late acting, affecting the development of wing muscles in adult
females (Fu et al. 2010). This has the benet that viable population reduction is not
seen until the adult stage, delaying the reduction in larval density and hence
maintaining high larval mortality rates for longer due to density-dependent compe-
tition of larvae in breeding sites (Black et al. 2011).
The rst eld trials of Ae. aegypti mosquitoes having the RIDL construct were
conducted using the OX513A strain in Malaysia and the Cayman Islands. In
Malaysia, Oxitec Ltd. and the Institute for Medical Research, Malaysia, worked
closely with the Malaysian government in conducting a risk assessment. Releases
were carried out in an uninhabited area to assess the mortality and dispersal
characteristics of released RIDL mosquitoes; however, negative reactions were
encountered from nongovernmental organizations and the media, preventing a trial
from being conducted in an inhabited area where the impact on wild Ae. aegypti
populations could be assessed (Enserink 2011).
In the Cayman Islands, Oxitec Ltd. worked with the local Mosquito Research and
Control Unit (MRCU), initially conducting smaller releases over the course of
4 weeks to assess the tness of genetically modied (GM) sterile males relative to
wild males and subsequently conducting a population suppression eld trial over the
course of several months, again using the OX513A Ae. aegypti strain. In a lab cage
study, GM sterile males were found to be more or less of equal competitiveness in
mating with wild females, and the lethality trait was found to be effective in all
crosses between GM sterile males and wild females (Harris et al. 2011). Subsequent
eld releases over a 4-week period found that GM males successfully mated with
wild females in the eld and fertilized their eggs resulting in unviable offspring;
however, the eld competitiveness of the GM males was estimated at ~56% that of
wild males, albeit with a very wide 95% condence interval of 3.2197% (Harris
et al. 2011).
The subsequent suppression eld trial in the Cayman Islands was carried out
across three contiguous areas on Grand Cayman island (denoted areas A, B, and C)
over a period of 23 weeks (Harris et al. 2012). The initial goal had been to achieve a
10:1 GM-to-wild male ratio by releasing across all three areas (55 ha in total);
however, production limitations led the actual achieved ratio to be signicantly
less (~2:1 GM-to-wild males), and a subsequent release in areas A and B still only
achieved a ratio of ~5:1 GM-to-wild males. The third phase of the release was
carried out solely in area A, achieving a release ratio of ~25:1 GM-to-wild males and
demonstrating the benet of a smaller trial area. Another benet of the area A release
was that area C served as a control and area B served as a buffer region. Signicant
population reduction was seen in this phase, with an 80% reduction in the mean
ovitrap index in area A relative to areas B and C over the last 7 weeks of the release
period (Harris et al. 2012).
Releases of GM sterile males in the Cayman Islands faced some controversy
(Nightingale 2010; Enserink 2010); however, the major criticisms concerned the
2 Field Trials of Gene Drive Mosquitoes: Lessons from Releases of Genetically... 25
manner in which information about the trials was disseminated, rather than the
conduct of the trials themselves. The releases did abide by national regulations, in
particular, a draft biosafety bill that had yet to become law, the MRCU obtained a
permit from the Cayman Islands Department of Agriculture, and a risk analysis and
environmental impact assessment were carried out. The degree of community
engagement was questioned; however, with several groups complaining, they had
not been given details of the releases in advance (Enserink 2010).
Subsequent releases in Brazil followed a much more transparent approach. From
the outset, a joint project was agreed, the Projeto Aedes Transgênico (PAT), between
the University of São Paulo and Oxitec Ltd. to explore the potential use of GM sterile
male Ae. aegypti as a form of urban mosquito control in terms of its social, technical,
and operational dimensions. The project was launched by Moscamed, a Brazilian
not-for-prot organization dependent on the Brazilian Ministry of Agriculture. The
project enjoyed signicant support in its early years as the government and public
were aware of dengue outbreaks caused by this mosquito, and governmental support
showed that they were being proactive in using the latest technology to control these
outbreaks. The PAT worked closely with the Brazilian regulatory system to obtain
required permits for eld activities and adopted a vigorous community engagement
campaign including school presentations, public events, interviews on TV and radio,
house visits, and involvement of the community in trap monitoring and surveillance
(de Campos et al. 2017).
The most well-documented trial of GM sterile male Ae. aegypti in Brazil was
carried out in the Itaberaba suburb of the city of Juazeiro in Bahia, Brazil. This site
had generally low socioeconomic indicators and relied on stored water to a large
extent, providing breeding sites for mosquitoes and leading to relatively high dengue
transmission. Similar to the Cayman Islands, the study area was divided into
treatment areas A and B and a control area, with treatment eventually being restricted
to area A in order to maintain sufciently high release ratios. A Moscamed mass-
rearing facility was built specically for the project, producing millions of GM
sterile males over the course of the study. Releases began with a range nder
phase lasting a little over a month, which allowed the release requirements to be
calibrated and estimates of parameters such as male mating competitiveness to be
rened. GM male mating competitiveness was estimated to be ~3.1% that of wild
males (95% CI: 2.53.6%), suggesting that releases for the suppressionphase
would need to be increased ninefold in order to achieve the target of 50% of mating
events involving a GM sterile male (Carvalho et al. 2015).
The GM sterile male eld trial in Brazil was successful, achieving a ~95%
reduction in mosquito density at the release site, albeit with large release require-
ments of ~140,000 mosquitoes per week over a 5.5 ha control site for ~3 months
(Carvalho et al. 2015). Enthusiasm for the GM sterile male approach was initially
raised when the Zika outbreak began in 2015; however, an unexpected complication
arose as untrue claims began to circulate in social media linking the Zika outbreak to
past releases of the GM mosquitoes (de Campos et al. 2017). This draws attention to
the importance of an enduring community engagement effort as well as political
engagement and stakeholder messaging.
26 J. M. Marshall and V. N. Vásquez
While this is not an invasive technology, these releases of sterile male mosquitoes
do provide lessons from which potential eld trials of gene drive mosquitoes may
learn. Releases both of chemosterilized Cx. quinquefasciatus in India and of GM
sterile Ae. aegypti in Brazil highlight the crucial importance of an effective and
sustained community engagement effort. This especially applies to technologies
developed in the Global North and applied in the Global South, which provide
much potential for community mistrust. Furthermore, releases of GM sterile Ae.
aegypti in both the Cayman Islands and Brazil highlight the importance of choosing
a study site in which the required release sizes can be achieved and in conducting a
range nder release phase to rene release requirements. For threshold-dependent
gene drive systems, this will be important to determine release sizes that exceed the
threshold, while for nonlocalized gene drive systems, this will be important to
determine release sizes that are expected to demonstrate population control within
the timeframe of the trial.
2.3 Lessons from the Wolbachia-based Incompatible Insect
A promising alternative to SIT and GM sterile male releases is IIT, in which male
mosquitoes are released that are infected with a Wolbachia strain absent from the
wild population, resulting in sterile matings with wild females that lack the
Wolbachia strain due to a phenomenon referred to as cytoplasmic incompatibility
(CI) (LePage et al. 2017) (Fig. 2.1). This strategy has proceeded with much less
resistance than GM approaches in recent years and serves as a case study for
potential releases of novel biological control technologies, particularly regarding
the use of factory rearing facilities (Zheng et al. 2019; Crawford et al. 2020).
The rst eld trial of IIT was conducted in Burma (now Myanmar) in 1967. The
technique was seen as an alternative to insecticide-based strategies given the grow-
ing insecticide resistance among target species, Cx. pipiens fatigans, a vector of
lymphatic lariasis (LF) which had proliferated in Southeast Asia at the time (Laven
1967). Despite successful elimination of the vector species from that trial site, the
approach has not been deployed operationally until recently due to concern that
accidental releases of Wolbachia-infected fertile females could result in the
Wolbachia strain spreading into the population, preventing further suppression
efforts. This is because Wolbachia is maternally inherited, and in most cases, the
only incompatible crosses are between infected males and uninfected females. In
2009 and 2010, however, subsequent trials were carried out in French Polynesia to
suppress populations of Aedes polynesiensis, a primary vector of LF in the South
Pacic(OConnor et al. 2012). Results from those eld experiments showed that
(1) Wolbachia-transfected Ae. polynesiensis males successfully competed for mates
following release and (2) the trial did not result in population replacement
2 Field Trials of Gene Drive Mosquitoes: Lessons from Releases of Genetically... 27
In the last few years, two factory-scale IIT projects have moved forward to
achieve community-scale mosquito population suppression: (1) an IIT program
supplemented with sterilizing irradiation (also termed IIT-SIT) in Guangzhou,
China (Zheng et al. 2019), and (2) an IIT program supplemented with factory-
scale automation of production and sex sorting in Fresno, California (Crawford
et al. 2020). The two projects represent different approaches to prevent population
replacement: (1) through greatly reducing the fertility of any Wolbachia-infected
females that may be accidentally released and (2) through using automation and
machine learning to reduce the number of accidentally released Wolbachia-infected
females effectively to zero.
In the IIT-SIT program in Guangzhou, Aedes albopictus, the main vector of
dengue and other arboviruses in Guangzhou, were generated having an articial
triple Wolbachia infection (termed HC), through the addition of the wPip Wolbachia
strain to the native double infection of the wAlbA and wAlbB strains of Wolbachia.
High levels of CI were conrmed such that matings of HC males with wild females
produced no viable offspring and maternal transmission of the triple Wolbachia
infection was conrmed, allowing efcient mass production of HC males. HC males
were exposed to low-dose irradiation at the pupal stage to reduce the fecundity of
any accidentally released HC females, and semi-eld cage studies conrmed that the
irradiated HC males effectively competed for mates leading to population suppres-
sion, without population replacement occurring due to released HC females. Fur-
thermore, as an additional safety precaution, HC females were shown to be less
competent at disease transmission than their wild counterparts (Zheng et al. 2019).
Fig. 2.1 (a) The use of Wolbachia as a means for both population suppression (incompatible insect
technique, IIT) and population replacement hinges on the inheritance pattern in which crosses
between Wolbachia-infected males and uninfected females produce unviable offspring due to
cytoplasmic incompatibility (CI), while crosses involving Wolbachia-infected females produce
Wolbachia-infected offspring due to Wolbachia being maternally inherited. (b) In IIT,
Wolbachia-infected males are released into a wild population lacking that strain of Wolbachia.
This leads to population suppression as mating events involving Wolbachia-infected males produce
no viable offspring. (c)InWolbachia-based population replacement, Wolbachia-infected females
are included in the release. This leads to population replacement as CI biases inheritance in favor of
Wolbachia when Wolbachia-infected females are present
28 J. M. Marshall and V. N. Vásquez
A trial carried out by the Wolbaki Biotech Company in 20162017 demonstrated
the high degree of population suppression possible when factory rearing of mosqui-
toes is involved. Irradiated HC males were released on a weekly basis on two
riverine islands within the jurisdiction of Guangzhou, with the ratio of released
HC to wild males varying between 8.7:1 and 15.8:1 over the 38-week intervention
period. Population suppression was highly successful, achieving a >94% reduction
in the number of hatched eggs per ovitrap, as compared to control sites, and an
8394% reduction in the number of wild adult females caught per trap. The success
of the program also led to a signicant increase in community support, with
interviews suggesting 13% of residents were supportive prior to the intervention
(notably, with 76% being neutral) and 54% were supportive following the interven-
tion (Zheng et al. 2019).
The IIT program in Fresno, CA, showcased the role that large-scale, automated
rearing and sex sorting of mosquitoes can play in increasing the scale of an IIT
intervention. In this case, Ae. aegypti, the main arboviral vector through much of the
Americas, was transfected with the wAlbB strain of Wolbachia, and sterility of
crosses between infected males and wild females was conrmed. An automated
larval rearing system was designed that, at maximum capacity, was able to produce
almost 3 million pupae per week. A multistep sex-separation process was then
designed that removed 95% of females at the pupal stage and the remainder at the
adult stage based on a machine learning algorithm informed by photographic images
as emerging adults walked down a narrow path. Estimates from the operation of this
system suggested that a single Wolbachia-infected female mosquito would be
released for every 900 million males, making the sex-sorting system near perfect
(Crawford et al. 2020).
A trial carried out through a partnership between the Debug Project of Verily Life
Sciences, MosquitoMate, and the Consolidated Mosquito Abatement District of
Fresno County in 2018 demonstrated dramatic population suppression over an
area nine times larger than that of the Guangzhou study. A total of more than
14 million Wolbachia-infected males were released as part of the study
(an average of more than 78,000 per day), which led to a 96% reduction in the
wild adult mosquito population; however, despite the large size of the releases,
elimination was not achieved, likely due to inward migration of wild mosquitoes
from neighboring untreated areas (Crawford et al. 2020). A public information
campaign was conducted around the trial; however, formal documentation of this
campaign is not yet available. A similar project is currently underway in Singapore
through a partnership between Verily Life Sciences and the National Environment
Agency of Singapore.
While neither a transgenic nor invasive technology, these IIT releases do provide
lessons regarding the scale of releases that can be achieved when investment is made
into automated rearing and sex-sorting facilities. Release requirements for
low-threshold gene drive mosquitoes will be orders of magnitude lower than those
for sterile male releases, and hence a facility capable of producing tens of millions of
mosquitoes, such as the one designed by Verily Life Sciences, would be capable of
achieving control over a much greater spatial scale than for IIT. The technological
2 Field Trials of Gene Drive Mosquitoes: Lessons from Releases of Genetically... 29
capacity for sex sorting is also encouraging given that male mosquitoes dont bite or
transmit diseases to humans and hence may also be preferable for gene drive
mosquito releases. The IIT releases enjoyed much less resistance from communities
and regulatory agencies than GM sterile male releases, despite acting through a
similar mechanism, highlighting issues that trials of gene drive mosquitoes will
likely also face and must invest in.
2.4 Lessons from Wolbachia-based Population Replacement
A second approach to the use of Wolbachia to control mosquito-borne disease
transmission is to intentionally include Wolbachia-infected females in a release. In
IIT, care is taken to only release Wolbachia-infected males, as CI causes matings
between Wolbachia-infected males and wild females to be sterile; however,
CI-induced sterility, combined with the fact that Wolbachia is maternally inherited,
provides an inheritance bias in favor of Wolbachia when Wolbachia-infected
females are also present (Turelli and Hoffmann 1991) (Fig. 2.1). For Wolbachia
strains that also block pathogen transmission, this can be used to drive the pathogen-
blocking trait into the mosquito population (Moreira et al. 2009). This strategy has
advanced signicantly over the last decade (Hoffmann et al. 2011) and, like IIT, has
faced much less resistance than GM strategies. It serves as an interesting case study
for potential releases of transgenic population replacement technologies, as it has
faced many of the non-GM issues that future gene drive programs will face.
The rst Wolbachia population replacement program was carried out by the
Eliminate Dengue project (now known as the World Mosquito Program) in the
communities of Yorkeys Knob and Gordonvale in Queensland, Australia (Hoffmann
et al. 2011). In this program, Ae. aegypti, the main vector of dengue and other
arboviruses in Queensland, was transfected with the wMel strain of Wolbachia from
Drosophila melanogaster, a strain that has been shown to (1) block dengue trans-
mission, (2) have a small associated tness cost, and (3) be capable of driving into a
small eld cage (Walker et al. 2011). Wolbachia displays threshold properties in the
presence of a tness cost such that releases above a certain population frequency
tend to spread, while releases below that frequency tend to be eliminated. The exact
value of the threshold is determined by the point at which the inheritance bias
induced by CI outweighs the tness cost associated with the infection and has
been estimated at ~2030% for the Wolbachia strain used in this release (Hoffmann
et al. 2011; Hancock et al. 2019).
The releases in Yorkeys Knob and Gordonvale were a clear successafter
10 weekly releases of 11,00022,000 Wolbachia-infected Ae. aegypti per week,
the Wolbachia infection reached near xation in both populations within 3 months,
despite a tropical storm postponing one of the releases in Gordonvale (Hoffmann
et al. 2011) (Fig. 2.2). The ner details of this program provide an excellent example
of how gene drive systems may be successfully trialed in the future. To begin, they
highlight the importance of a detailed monitoring effort and adaptive release
30 J. M. Marshall and V. N. Vásquez
protocol. The releases in Yorkeys Knob and Gordonvale were accompanied by a
network of 29 Biogents Sentinel mosquito traps that monitored Wolbachia infection
frequency at the block level. Heterogeneity in Wolbachia infection frequency was
00 250 500
0 50 100
Time (days) Time (days)
Wolbachia frequency
Drive allele frequency
Number of releases
Field observations
Model predictions
145.700 145.705
~0.533 m
145.710 145.715 145.720 145.725
Fig. 2.2 (a) Landscape of Yorkeys Knob and Trinity Park in Queensland, Australia, where eld
trials of Wolbachia-based population replacement for Aedes aegypti were carried out and where
trials of reciprocal chromosomal translocations were simulated. (b) Blue lines depict data for
Wolbachia frequency over time from the Wolbachia population replacement eld trial conducted
in Yorkeys Knob in 2011 (Hoffmann et al. 2011), with line thickness representing 95% binomial
condence intervals around observed proportions. Red lines depict simulated data for an analogous
release scheme consisting of 20 Wolbachia-infected mosquitoes per household at a coverage of
30% over 10 weeks, demonstrating good agreement with eld data (Sánchez et al. 2020). (c)
Translocation frequency over time for a given number of weekly releases of 20 adult male Ae.
aegypti mosquitoes homozygous for the translocation per household with the intent of population
replacement in Yorkeys Knob. Results are depicted for a coverage of 50%, at which seven or more
releases result in the translocation being driven into the population (Sánchez et al. 2020). Due to the
50% threshold property of translocations, the same release scheme for wild types can be used to
remediate translocations from the population
2 Field Trials of Gene Drive Mosquitoes: Lessons from Releases of Genetically... 31
observed, and releases were supplemented in areas where Wolbachia frequency was
Monitoring for unintended spread outside the study area was also conducted, and
this did indeed reveal limited long-distance spread into a neighboring suburb from
Yorkeys Knob and across a freeway from Gordonvale (Hoffmann et al. 2011).
Although these migrants were expected to be lost due to being present at subthresh-
old levels, continued monitoring was important to conrm this. Continued monitor-
ing was also conducted at the trials sites to conrm enduring intervention efcacy,
and while the Wolbachia infection remained at near xation for several years
following the release, a low frequency of uninfected mosquitoes has also persisted,
likely due to immigration (Hoffmann et al. 2014).
The releases in Yorkeys Knob and Gordonvale also highlight the importance of
preparing for unexpected events. In addition to the tropical storm that affected both
release sites and postponed one of the releases in Gordonvale, releases in a portion of
Yorkeys Knob ceased two-thirds of the way into the intervention following a
reported dengue case (Hoffmann et al. 2011). Although this dengue case likely
originated elsewhere, a reactive insecticide intervention was carried out in surround-
ing households in agreement with local disease control protocols. Trials of mosqui-
toes with gene drive systems should make allowances for events such as these.
Encouragingly, the Wolbachia infection continued to spread through the Yorkeys
Knob Ae. aegypti population despite this, and no secondary dengue cases were
documented following the reported case.
The Yorkeys Knob and Gordonvale releases provide an example of a successful
community and regulatory engagement process. Community engagement was car-
ried out over 2 years leading up to the releases and consisted of informal interviews,
semi-structured in-depth interviews, qualitative and quantitative surveys, focus
groups, historical research, and face-to-face presentations at community meetings
(Hoffmann et al. 2011; McNaughton 2012). Issues explored through these activities
included the sociopolitical context, lay knowledge of dengue fever and biological
control programs, and acceptability of the project. Community members did raise
concerns about a previous local biological control programthe introduction of the
cane toad near Gordonvale in the 1930s. Largely seen as a failed biological control
program, this was raised as a cautionary tale indicating the limits of scientic
knowledge and the unpredictability of ecological interventions (McNaughton 2012).
The Queensland releases enjoyed substantial community support, with 85% of
respondents viewing Wolbachia as an acceptable dengue prevention strategy in a
March 2010 telephone survey (ahead of insecticides, at 66% acceptance) and 84% of
respondents stating they would support a release that they were informed and
updated about, that had regulatory oversight, and that was shown to be safe for
people and the environment by a risk assessment carried out by Australias Com-
monwealth Scientic and Industrial Research Organisation (CSIRO) (McNaughton
2012). The releases were ultimately approved by the Australian Pesticides and
Veterinary Medicines Authority (APVMA) following risk assessments by CSIRO
(Murphy et al. 2010) and the APVMA with support from the Federal Common-
wealth Governments Department of the Environment, Water, Heritage and the Arts
32 J. M. Marshall and V. N. Vásquez
(Marshall 2011). The World Mosquito Program is now exploring application of their
technology beyond Australia, with active collaborations throughout Latin America,
Asia, and Oceania.
In summary, key lessons from the Wolbachia-based population replacement
strategy include the importance of (1) a detailed monitoring protocol to assess
heterogeneity of spread at the eld site, (2) an adaptive release scheme to supplement
releases in areas of low Wolbachia frequency, (3) additional monitoring to assess
levels of unintended spread to neighboring areas, and (4) preparing for unexpected
events. The fact that Wolbachia infection has the potential to persist in the mosquito
population for extended periods, and perhaps indenitely, also emphasizes the need
for a long-term, comprehensive, and multifaceted community engagement program.
2.5 Considerations for Trials with Reciprocal
Chromosomal Translocations
Lessons from eld trials of Wolbachia-based population replacement systems apply
most closely to threshold-dependent gene drive systems, which are also expected to
spread if released above a certain threshold frequency and to be eliminated if present
below that frequency. One of the rst of these systems to be proposed (Serebrovskii
1940; Curtis 1968), and perhaps currently one of the most promising (Sánchez et al.
2020), is reciprocal chromosomal translocations. These result from a mutual
exchange between terminal segments of two nonhomologous chromosomes and
produce a heterozygote reproductive disadvantage because, when translocation
heterozygotes mate, several crosses result in unbalanced genotypes and hence
unviable offspring. This produces a threshold frequency of 50%, which increases
in the presence of a tness cost (Curtis 1968). Early attempts to generate trans-
locations through radiation-induced mutagenesis were abandoned due to high asso-
ciated tness costs (Laven et al. 1972; Lorimer et al. 1972); however, interest has
been reignited as site-specic translocations have recently been generated using
CRISPR (Lekomtsev et al. 2016; Jiang et al. 2016), and translocations generated
in D. melanogaster using endonucleases were recently shown to drive in laboratory
experiments with a threshold frequency of ~50% (Buchman et al. 2018).
A recent modeling study suggests that translocations represent one of the best
systems to implement in eld trials due to their symmetrical threshold properties and
strong connement potential. A key advantage of translocations is that releases
required to introduce them into a population are of a similar magnitude to wild-
type releases required to eliminate them once they have been introduced (Sánchez
et al. 2020). Population replacement and reversion were modeled at the household
level in the suburb of Yorkeys Knob, the site of the Wolbachia population replace-
ment study, with low levels of migration modeled to the neighboring suburb of
Trinity Park in Queensland, Australia (Fig. 2.2). Population replacement could be
achieved in simulations with seven or more weekly releases of 20 Ae. aegypti males
2 Field Trials of Gene Drive Mosquitoes: Lessons from Releases of Genetically... 33
homozygous for the translocations per household per week (a similar magnitude to
that used in the Wolbachia population replacement trial at the same site) and for a
coverage of 50% of the households in the community. Elimination could be achieved
for the same release scheme using wild Ae. aegypti mosquitoes.
One benet of translocations, and other underdominant systems that have a
threshold in the absence of a tness cost, is that their release threshold is more
robust than that for Wolbachia, which only arises in the presence of a tness cost.
This property leads to translocations being more robustly conneable to a eld site
than a Wolbachia infection, since they are unlikely to exceed the release threshold in
a neighboring population purely through migration, even if they spread to near
xation at the trial site. In the translocation modeling study in Yorkeys Knob and
Trinity Park (Sánchez et al. 2020), it was considered unlikely that Ae. aegypti
mosquitoes would travel from one suburb to another by their own ight, especially
in numbers sufcient to exceed the release threshold there, and so batch migration
was instead considered, in which several mosquitoes are carried, perhaps by a
vehicle, from one suburb to another at once. Batch migration events were modeled
as occurring between randomly chosen neighborhoods, and the number of daily
migration events and effective number of adults carried per event were varied.
Results from this modeling study made a strong case for the potential to conne
translocations to the release site, as the number of daily migration events required for
the translocation to exceed the threshold in the neighboring suburb exceeded those
inferred from eld data. Specically, 34 daily migration events consisting of
batches of ten adults were required for translocations to spillover to the neighboring
suburb in simulations (Sánchez et al. 2020), while eld data suggested 12 daily
migration events consisting of batches of less than ve adult females (Hoffmann
et al. 2011).
Collectively, these modeling results for translocations are encouraging for the
potential to conduct eld trials of Ae. aegypti mosquitoes with translocations
because (1) translocations could be introduced on a suburban scale and remediated
through releases of non-disease-transmitting male mosquitoes with release sizes on
the scale of what has been previously implemented and (2) spillover of transloca-
tions into neighboring suburbs is unlikely. Lessons for the conduct of eld trials with
translocations may be drawn from the eld trials previously described in this
chaptermost importantly, for Wolbachia-based population replacement. These
lessons highlight the importance of a detailed monitoring effort, including outside
the study area, and of an adaptive release protocol that can respond to heterogeneities
in spread at the trial site. They also highlight the importance of preparing for
unexpected events and for conducting a long-term and comprehensive community
engagement program, given that translocations have the potential to persist in the
environment long term. A comparison of the RIDL and IIT releases suggests that
community engagement and regulatory requirements for translocations may be
stricter than for those for Wolbachia due to the fact that mosquitoes with trans-
locations, generated using CRISPR or other endonucleases, will be considered GM
organisms. Finally, regarding the release protocol, including a range nder release
34 J. M. Marshall and V. N. Vásquez
phase may help to rene tness cost estimates and release requirements for trans-
locations, as per a lesson from the RIDL eld trial in Brazil.
2.6 Considerations for Trials with CRISPR-based Gene
Drive Systems
Finally, lessons from the eld trials discussed here have implications for the spec-
trum of CRISPR-based gene drives, from those that are nonlocalized to those that are
self-limiting. Recent attention has focused on CRISPR-based homing gene drives,
for their ability to spread widely and their potential to control vector-borne diseases
on a wide scale (Gantz et al. 2015; Kyrou et al. 2018); however, there are also
threshold-dependent gene drive systems that can now be engineered using CRISPR,
such as chromosomal translocations (Buchman et al. 2018) and various forms of
underdominance (Akbari et al. 2013), as well as temporally self-limiting gene drive
systems, such as split drive (Li et al. 2020), which display transient drive activity
before being eliminated by virtue of a tness cost. The CRISPR revolution has also
enabled gene drive countermeasures to be engineered, such as homing-based drive
remediation systems, ERACR (element for the reversal of the autocatalytic chain
reaction) and e-CHACR (erasing construct hitchhiking on the autocatalytic chain
reaction) (Gantz and Bier 2016; Xu et al. 2020).
CRISPR-based homing gene drive systems bias inheritance in their favor by
cleaving a highly specic target sequence in the host genome and copying them-
selves to the cut chromosome through a mechanism known as homology-directed
repair (Gantz and Bier 2015; Champer et al. 2016). For high homing efciencies and
low-to-moderate tness costs, these systems are capable of driving into populations
from arbitrarily low initial frequencies. This property allows them to spread widely,
and hence they are considered nonlocalized.For these gene drive systems, while
we may learn from eld trials of Wolbachia-based population replacement systems,
the scale of their potential spread and impact leads to additional and unique chal-
lenges that we must carefully consider.
One way to manage the risks associated with the potential wide-scale spread of
homing-based gene drive systems is for testing to proceed iteratively through
multiple phases, with each phase involving a larger spatial scale and a higher degree
of human or environmental exposure (James et al. 2018) (Fig. 2.3). In this phased
release pathway, initial studies are to be conducted in contained laboratories and
Fig. 2.3 Phased release pathway for CRISPR-based homing gene drive systems
2 Field Trials of Gene Drive Mosquitoes: Lessons from Releases of Genetically... 35
insectaries, where product efcacy and safety are studied. Entering eld-testing is a
big decision, given the anticipated difculty of remediating a homing-based gene
drive system that is capable of spreading widely. Large outdoor cages present one
option for moving beyond the laboratory; however, this is not considered essential
since some mosquito behaviors, such as mating, and parameters, such as tness, can
only be meaningfully studied in the eld. Furthermore, studies in outdoor cages must
anticipate the possibility of escape occurring, and hence similar safety and efcacy
criteria must be met before either outdoor cage studies or small-scale isolated
releases are performed. Initial outdoor testing should be conducted at eld sites
within which the gene drive system is expected to be contained, for instance, on
oceanic islands, following which open releases would be conducted on iteratively
larger spatial scales (James et al. 2018).
Another consideration for trials of nonlocalized gene drive systems is that
regulators are likely to require that a remediation plan be in place prior to eld-
testing (James et al. 2018). The chosen remediation strategy will depend on a
number of factors, including the mode of action of the drive system and the scale
and geography of the eld site. A default remediation plan would be a large-scale
insecticide-based campaign to eliminate the vector population at the eld site. This
would require an assessment of insecticide resistance in the local vector population
prior to the gene drive trial. Failing this, releases of non-disease-transmitting male
mosquitoes carrying a drive-resistant allele that restores the function of the gene
targeted by the drive system are an attractive option, especially if the drive-resistant
allele is sourced from a wild population.
Gene drive countermeasures such as ERACR and e-CHACR are another option
for remediation. The ERACR system consists of a second homing system with a
target site corresponding to the original drive system, essentially removing the
original system as it homes into it, while utilizing the Cas9 of the original system
and thus removing that as well (Gantz and Bier 2016; Xu et al. 2020). The e-CHACR
system uses the Cas9 from the original homing system to home into a second site in
the genome in addition to the site of the original drive system, thus driving itself into
the population while removing the original system and its Cas9 in the process (Gantz
and Bier 2016, Xu et al. 2020). Both of these systems hold promise; but they may not
be the rst choice for remediation efforts as they introduce additional transgenes into
populations from which transgenes are trying to be removed.
Another potential phased release pathway is to precede the release of a
nonlocalized gene drive system with a self-limiting one. Ideally, such a release
would provide insights into the expected behavior of the nonlocalized system, and
hence there should be strong resemblance between the two systems, to the extent
possible. For a CRISPR-based homing gene drive system, one possibility is to begin
with a trial of a split drive system, in which the Cas9 and guide RNA components are
separated at different loci (Li et al. 2020). In the split drive system, transient drive
activity occurs at the guide RNA locus when the Cas9 and guide RNA alleles
co-occur in an organism; however, the Cas9 allele is gradually eliminated from the
population due to its tness cost, followed by the guide RNA if it also has a tness
cost. This transient drive activity also leads to spatial connement, since a gene can
36 J. M. Marshall and V. N. Vásquez
only disperse so far in a limited number of generations. Intermediate technologies
also exist for other systems. For instance, a driving Y chromosome that spreads by
cleaving the X chromosome at multiple sites during spermatogenesis is expected to
spread on a wide scale (Galizi et al. 2014); however, if linked to an autosome, it is
self-limiting, providing an opportunity for intermediate study in the eld.
For self-limiting CRISPR-based gene drive systems that could be used as an
intermediate system in a eld trial, similar eld trial considerations apply as for
chromosomal translocations. Namely, the ability to conne the release to the trial
site, and to remediate transgenes from the environment as needed, is a great strength.
Furthermore, it is important to combine a detailed monitoring effort, both in and
outside the trial site, with an adaptive release protocol to respond to heterogeneities
in spread, and to make allowances for unexpected events. A range nder release
phase may help to rene tness cost estimates and release requirements.
For nonlocalized CRISPR-based gene drive systems, the potentially wide scale of
spread and difculty of remediation emphasize the need to monitor for the gene drive
system both in and outside the eld trial area. Additionally, a range nder release
phase may help to predict release schemes capable of achieving population control
within the desired timeframe. Finally, as the spatial scale of the release grows,
lessons may be learned from the experience of the Fresno IIT trial regarding
automated rearing and sex sorting of mosquitoes. Knowledge of the potential scale
of mosquito production will allow us to set expectations for wide-scale vector-borne
disease control.
As for all of the systems discussed in this chapter, effective community and
regulatory engagement is essential prior to eld trials of mosquitoes engineered with
CRISPR-based gene drive systems; however, this is especially important for trials of
nonlocalized gene drive systems. Mosquitoes engineered with these systems are GM
organisms capable of spreading widely, potentially across international borders, and
are often developed in the Global North for application in the Global South. Their
potential to spread across international borders highlights the desirability of a
multicountry or regional agreement on their release, especially when a country that
shares a border with another is being considered for eld trials. Indeed, such
agreements may be required by the Cartagena Protocol on Biosafety, which governs
the safe transfer, handling, and use of GM organisms (referred to as living modied
organismsin the protocol), including their transboundary spread (Secretariat of the
Convention on Biological Diversity 2000; Marshall 2010).
2.7 Conclusion
The limitations of traditional insecticide-based strategies to control mosquito
populations, and, in particular, the widespread emergence of insecticide resistance,
have spurred interest in a variety of novel biological and genetics-based vector
control strategies, including SIT, IIT, RIDL, Wolbachia-based population replace-
ment, and CRISPR-based gene drive (Benelli et al. 2016). Trials of RIDL, IIT, and
2 Field Trials of Gene Drive Mosquitoes: Lessons from Releases of Genetically... 37
Wolbachia over the last decade provide a series of case studies from which we may
learn in preparing for eld trials of CRISPR-based gene drive systems (Table 2.2).
There are challenges associated with gene drive technologiesnotably, the
controversies surrounding GM organisms and the potential for spread across inter-
national borders. However, these challenges are also a reason for promise as half of
the worlds population is at risk of vector-borne diseases, and genetic engineering
provides new opportunities to interfere with pathogen transmission. In learning from
recent eld trials, we seek to move these technologies forward carefully and respon-
sibly toward the eventual goal of global vector-borne disease control.
Table 2.2 Signicant eld trials of novel biological and genetics-based mosquito control strategies
Method Species Location Year Outcome Reference
SIT Anopheles
1962 Poor mating
et al. (1962)
IIT Culex pipiens
Burma (now
1967 Successful
SIT Culex
India 1971
Singh et al.
SIT Anopheles
El Salvador 1971
Lowe et al.
RIDL Aedes aegypti Cayman
2009 Small-scale
Harris et al.
IIT Aedes
of efcacy
et al. (2012)
Wolbachia popu-
Ae. aegypti Queensland,
2011 Successful popu-
et al. (2011)
RIDL Ae. aegypti Juazeiro,
et al. (2015)
RIDL Ae. aegypti Jacobina,
2013 Suppression and
et al. (2017)
IIT Aedes albopictus Kentucky,
2014 Signicant
Mains et al.
IIT-SIT Ae. albopictus Guangzhou,
Zheng et al.
IIT Ae. aegypti California,
et al. (2020)
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2 Field Trials of Gene Drive Mosquitoes: Lessons from Releases of Genetically... 41
... Systems have also been designed to either turn on or turn off GD activity in the presence or absence of small organic molecules that can easily enter cells [50,51]. While reversal and inducible GD systems hold promise, developers themselves caution against unforeseen consequences [49], and indicate that reversal GDs may not necessarily be the first choice for remediation efforts due to the associated uncertainty of introducing another GD approach [52]. ...
... Currently, there is limited direct experience in conducting the risk assessment of GDMI releases. Nonetheless, principles and methodologies for risk assessment and risk management, experience from the risk assessment of GMIs that do not contain engineered GDs, and knowledge from other disease vector and pest control strategies, are relevant to performing GDMI risk assessments [3,15,52]. For example, there is substantial experience with releasing insects for genetic and biological disease vector/pest control, including their risk assessment and post-release monitoring (where applicable), from which lessons can be learnt. ...
... Since self-sustaining engineered GDs are designed for widespread and long-standing control, spatially and/or temporally restricting their spread/persistence would not necessarily be in line with the intended outcome of their release. Therefore, besides gathering data under confined conditions, developers and potential applicants may wish to consider the utility of prior field testing of a related self-limiting strain (note however, that there should be a strong resemblance between the two systems, to the extent possible) as an intermediate step to gather evidence for model development and refinement, and reduce uncertainties in risk assessment [15,16,52]. ...
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Potential future application of engineered gene drives (GDs), which bias their own inheritance and can spread genetic modifications in wild target populations, has sparked both enthusiasm and concern. Engineered GDs in insects could potentially be used to address long-standing challenges in control of disease vectors, agricultural pests and invasive species, or help to rescue endangered species, and thus provide important public benefits. However, there are concerns that the deliberate environmental release of GD modified insects may pose different or new harms to animal and human health and the wider environment, and raise novel challenges for risk assessment. Risk assessors, risk managers, developers, potential applicants and other stakeholders at many levels are currently discussing whether there is a need to develop new or additional risk assessment guidance for the environmental release of GD modified organisms, including insects. Developing new or additional guidance that is useful and practical is a challenge, especially at an international level, as risk assessors, risk managers and many other stakeholders have different, often contrasting, opinions and perspectives toward the environmental release of GD modified organisms, and on the adequacy of current risk assessment frameworks for such organisms. Here, we offer recommendations to overcome some of the challenges associated with the potential future development of new or additional risk assessment guidance for GD modified insects and provide considerations on areas where further risk assessment guidance may be required.
... Systems have also been designed to either turn on or turn off gene drive activity in the presence or absence of small organic molecules that can easily enter cells (Heffel and Finnigan, 2019;López Del Amo et al., 2020). While reversal and inducible engineered gene drive systems hold promise for risk management, developers themselves caution against unforeseen consequences (Xu et al., 2020), and indicate that reversal engineered gene drives may not necessarily be the first choice for remediation efforts due to the associated uncertainty of introducing another gene drive approach (Marshall and Vasquez, 2021). ...
... Implementing a modular, phased approach to authorisations for nonlocalised and self-sustaining engineered gene drives remains conceptual at present, in part due to their current inability for recall. Therefore, developers and potential applicants may wish to consider the utility of prior field testing of a closely related self-limiting and/or localised strain as an intermediate step to gather evidence for model development and refinement, and reduce uncertainty in risk assessment (James et al., 2018;EFSA et al., 2020a;Marshall and Vasquez, 2021;WHO, 2021). Self-limiting and/or localisation approaches could be used as biological or molecular risk mitigation strategies to limit the spread and/or persistence of engineered gene drives. ...
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The ability to engineer gene drives (genetic elements that bias their own inheritance) has sparked enthusiasm and concerns. Engineered gene drives could potentially be used to address long-standing challenges in the control of insect disease vectors, agricultural pests and invasive species, or help to rescue endangered species. However, risk concerns and uncertainty associated with potential environmental release of gene drive modified insects (GDMIs) have led some stakeholders to call for a global moratorium on such releases or the application of other strict precautionary measures to mitigate perceived risk assessment and risk management challenges. Instead, we provide recommendations that may help to improve the relevance of risk assessment and risk management frameworks for environmental releases of GDMIs. These recommendations include: (1) developing additional and more practical risk assessment guidance to ensure appropriate levels of safety; (2) making policy goals and regulatory decision-making criteria operational for use in risk assessment so that what constitutes harm is clearly defined; (3) ensuring a more dynamic interplay between risk assessment and risk management to manage uncertainty through closely interlinked pre-release modelling and post-release monitoring; (4) considering potential risks against potential benefits, and comparing them with those of alternative actions to account for a wider (management) context; and (5) implementing a modular, phased approach to authorisations for incremental acceptance and management of risks and uncertainty. Along with providing stakeholder engagement opportunities in the risk analysis process, the recommendations proposed may enable risk managers to make choices that are more proportionate and adaptive to potential risks, uncertainty and benefits of GDMI applications, and socially robust.
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The discovery of CRISPR-based gene editing and its application to homing-based gene drive systems has been greeted with excitement, for its potential to control mosquito-borne diseases on a wide scale, and concern, for the invasiveness and potential irreversibility of a release. Gene drive systems that display threshold-dependent behavior could potentially be used during the trial phase of this technology, or when localized control is otherwise desired, as simple models predict them to spread into partially isolated populations in a confineable manner, and to be reversible through releases of wild-type organisms. Here, we model hypothetical releases of two recently engineered threshold-dependent gene drive systems-reciprocal chromosomal translocations and a form of toxin-antidote-based underdominance known as UD MEL-to explore their ability to be confined and remediated. We simulate releases of Aedes aegypti, the mosquito vector of dengue, Zika, and other arboviruses, in Yorkeys Knob, a suburb of Cairns, Australia, where previous biological control interventions have been undertaken on this species. We monitor spread to the neighboring suburb of Trinity Park to assess confinement. Results suggest that translocations could be introduced on a suburban scale, and remediated through releases of non-disease-transmitting male mosquitoes with release sizes on the scale of what has been previously implemented. UD MEL requires fewer releases to introduce, but more releases to remediate, including of females capable of disease transmission. Both systems are expected to be confineable to the release site; however, spillover of translocations into neighboring populations is less likely. Our analysis supports the use of translocations as a threshold-dependent drive system capable of spreading disease-refractory genes into Ae. aegypti populations in a confineable and reversible manner. It also highlights increased release requirements when incorporating life history and population structure into models. As the technology nears implementation, further ecological work will be essential to enhance model predictions in preparation for field trials.
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The range of the mosquito Aedes aegypti continues to expand, putting more than two billion people at risk of arboviral infection. The sterile insect technique (SIT) has been used to successfully combat agricultural pests at large scale, but not mosquitoes, mainly because of challenges with consistent production and distribution of high-quality male mosquitoes. We describe automated processes to rear and release millions of competitive, sterile male Wolbachia-infected mosquitoes, and use of these males in a large-scale suppression trial in Fresno County, California. In 2018, we released 14.4 million males across three replicate neighborhoods encompassing 293 hectares. At peak mosquito season, the number of female mosquitoes was 95.5% lower (95% CI, 93.6–96.9) in release areas compared to non-release areas, with the most geographically isolated neighborhood reaching a 99% reduction. This work demonstrates the high efficacy of mosquito SIT in an area ninefold larger than in previous similar trials, supporting the potential of this approach in public health and nuisance-mosquito eradication programs. Mosquitoes are nearly eradicated in three suburbs of California using accurately sorted sterile male mosquitoes.
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Aedes aegypti, the principal mosquito vector for many arboviruses that increasingly infect millions of people every year. With an escalating burden of infections and the relative failure of traditional control methods, the development of innovative control measures has become of paramount importance. The use of gene drives has sparked significant enthusiasm for genetic control of mosquitoes; however, no such system has been developed in Ae. aegypti. To fill this void, here we develop several CRISPR-based split gene drives for use in this vector. With cleavage rates up to 100% and transmission rates as high as 94%, mathematical models predict that these systems could spread anti-pathogen effector genes into wild populations in a safe, confinable and reversible manner appropriate for field trials and effective for controlling disease. These findings could expedite the development of effector-linked gene drives that could safely control wild populations of Ae. aegypti to combat local pathogen transmission.
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The radiation-based sterile insect technique (SIT) has successfully suppressed field populations of several insect pest species, but its effect on mosquito vector control has been limited. The related incompatible insect technique (IIT)—which uses sterilization caused by the maternally inherited endosymbiotic bacteria Wolbachia—is a promising alternative, but can be undermined by accidental release of females infected with the same Wolbachia strain as the released males. Here we show that combining incompatible and sterile insect techniques (IIT–SIT) enables near elimination of field populations of the world’s most invasive mosquito species, Aedes albopictus. Millions of factory-reared adult males with an artificial triple-Wolbachia infection were released, with prior pupal irradiation of the released mosquitoes to prevent unintentionally released triply infected females from successfully reproducing in the field. This successful field trial demonstrates the feasibility of area-wide application of combined IIT–SIT for mosquito vector control.
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In the human malaria vector Anopheles gambiae, the gene doublesex (Agdsx) encodes two alternatively spliced transcripts, dsx-female (AgdsxF) and dsx-male (AgdsxM), that control differentiation of the two sexes. The female transcript, unlike the male, contains an exon (exon 5) whose sequence is highly conserved in all Anopheles mosquitoes so far analyzed. We found that CRISPR–Cas9-targeted disruption of the intron 4–exon 5 boundary aimed at blocking the formation of functional AgdsxF did not affect male development or fertility, whereas females homozygous for the disrupted allele showed an intersex phenotype and complete sterility. A CRISPR–Cas9 gene drive construct targeting this same sequence spread rapidly in caged mosquitoes, reaching 100% prevalence within 7–11 generations while progressively reducing egg production to the point of total population collapse. Owing to functional constraint of the target sequence, no selection of alleles resistant to the gene drive occurred in these laboratory experiments. Cas9-resistant variants arose in each generation at the target site but did not block the spread of the drive.
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Recent reports have suggested that self-propagating CRISPR-based gene drive systems are unlikely to efficiently invade wild populations due to drive-resistant alleles that prevent cutting. Here we develop mathematical models based on existing empirical data to explicitly test this assumption for population alteration drives. Our models show that although resistance prevents spread to fixation in large populations, even the least effective drive systems reported to date are likely to be highly invasive. Releasing a small number of organisms will often cause invasion of the local population, followed by invasion of additional populations connected by very low rates of gene flow. Hence, initiating contained field trials as tentatively endorsed by the National Academies report on gene drive could potentially result in unintended spread to additional populations. Our mathematical results suggest that self-propagating gene drive is best suited to applications such as malaria prevention that seek to affect all wild populations of the target species.
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Gene drive technology offers the promise for a high-impact, cost-effective, and durable method to control malaria transmission that would make a significant contribution to elimination. Gene drive systems, such as those based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein, have the potential to spread beneficial traits through interbreeding populations of malaria mosquitoes. However, the characteristics of this technology have raised concerns that necessitate careful consideration of the product development pathway. A multidisciplinary working group considered the implications of low-threshold gene drive systems on the development pathway described in the World Health Organization Guidance Framework for testing genetically modified (GM) mosquitoes, focusing on reduction of malaria transmission by Anopheles gambiae s.l. mosquitoes in Africa as a case study. The group developed recommendations for the safe and ethical testing of gene drive mosquitoes, drawing on prior experience with other vector control tools, GM organisms, and biocontrol agents. These recommendations are organized according to a testing plan that seeks to maximize safety by incrementally increasing the degree of human and environmental exposure to the investigational product. As with biocontrol agents, emphasis is placed on safety evaluation at the end of physically confined laboratory testing as a major decision point for whether to enter field testing. Progression through the testing pathway is based on fulfillment of safety and efficacy criteria, and is subject to regulatory and ethical approvals, as well as social acceptance. The working group identified several resources that were considered important to support responsible field testing of gene drive mosquitoes.
CRISPR-Cas9-based gene drive systems possess the inherent capacity to spread progressively throughout target populations. Here we describe two self-copying (or active) guide RNA-only genetic elements, called e-CHACRs and ERACRs. These elements use Cas9 produced in trans by a gene drive either to inactivate the cas9 transgene (e-CHACRs) or to delete and replace the gene drive (ERACRs). e-CHACRs can be inserted at various genomic locations and carry two or more gRNAs, the first copying the e-CHACR and the second mutating and inactivating the cas9 transgene. Alternatively, ERACRs are inserted at the same genomic location as a gene drive, carrying two gRNAs that cut on either side of the gene drive to excise it. e-CHACRs efficiently inactivate Cas9 and can drive to completion in cage experiments. Similarly, ERACRs, particularly those carrying a recoded cDNA-restoring endogenous gene activity, can drive reliably to fully replace a gene drive. We compare the strengths of these two systems.
A promising strategy for reducing the transmission of dengue and other arboviral human diseases by Aedes aegypti mosquito vector populations involves field introductions of the endosymbiotic bacteria Wolbachia. Wolbachia infections inhibit viral transmission by the mosquito, and can spread between mosquito hosts to reach high frequencies in the vector population. Wolbachia spreads by maternal transmission, and spread dynamics can be variable and highly dependent on natural mosquito population dynamics, population structure and fitness components. We develop a mathematical model of an Ae. aegypti metapopulation that incorporates empirically validated relationships describing density‐dependent mosquito fitness components. We assume that density dependent relationships differ across subpopulations, and construct heterogeneous landscapes for which model‐predicted patterns of variation in mosquito abundance and demography approximate those observed in field populations. We then simulate Wolbachia release strategies similar to that used in field trials. We show that our model can produce rates of spatial spread of Wolbachia similar to those observed following field releases. We then investigate how different types of spatio‐temporal variation in mosquito habitat, as well as different fitness costs incurred by Wolbachia on the mosquito host, influence predicted spread rates. We find that fitness costs reduce spread rates more strongly when the habitat landscape varies temporally due to stochastic and seasonal processes. Synthesis and applications:Our empirically based modelling approach represents effects of environmental heterogeneity on the spatial spread of Wolbachia. The models can assist in interpreting observed spread patterns following field releases and in designing suitable release strategies for targeting spatially heterogeneous vector populations. This article is protected by copyright. All rights reserved.
Replacement of wild insect populations with transgene-bearing individuals unable to transmit disease or survive under specific environmental conditions using gene drive provides a self-perpetuating method of disease prevention. Mechanisms that require the gene drive element and linked cargo to exceed a high threshold frequency in order for spread to occur are attractive because they offer several points of control: they bring about local, but not global population replacement; and transgenes can be eliminated by reintroducing wildtypes into the population so as to drive the frequency of transgenes below the threshold frequency required for drive. Reciprocal chromosome translocations were proposed as a tool for bringing about high threshold population replacement in 1940 and 1968. However, translocations able to achieve this goal have only been reported once, in the spider mite Tetranychus urticae, a haplo-diploid species in which there is strong selection in haploid males for fit homozygotes. We report the creation of engineered translocation-bearing strains of Drosophila melanogaster, generated through targeted chromosomal breakage and homologous recombination. These strains drive high threshold population replacement in laboratory populations. While it remains to be shown that engineered translocations can bring about population replacement in wild populations, these observations suggest that further exploration of engineered translocations as a tool for controlled population replacement is warranted.