1. Genetic control of mosquitoes
HEALTHY PEOPLE HEALTHY ENVIRONMENT
Genetic control of mosquitoes
Luke Alphey, Founder and Chief Scientific Officer
Visiting Professor in Zoology, University of Oxford

2. Injecting DNA into mosquito eggs
Oxitec Ltd
Based in Oxford, UK, 30 employees
Founded in 2002 to commercialise new technology from Oxford University
Control of insects
Agricultural pests
Public Health (mosquitoes)
Injecting DNA into mosquito eggs

3. Introduction Why? How? When?
Pest insects cause $$bn damage and transmit major diseases
How?
Engineered sterile males
RIDL®: Release of Insects carrying a Dominant Lethal genetic system
Genetics, molecular biology
When?
Initial strains successfully tested in field
Marker-only moth: USA 2006
RIDL mosquitoes: Cayman Islands 2009, Malaysia 2010, Brazil 2011

4. Increased Risk of Vector Borne Disease
Dengue - growing global pandemic
Over 100 million cases annually
Severity increasing
No specific medication or vaccine
Chikungunya - emerging threat
1.8 million cases in last 6 years (WHO)
Reunion (2005) had 266,000 cases and 254 deaths
Italy (2007) – 197 cases and I death
Oxitec/Nimmo 4

5. WHO
Dengue control

6. Male mosquitoes! In fact the sound I played you at the beginning was the love song of two courting mosquitoes. Males are highly evolved to find females.
And when they do, in the normal way of things, you get lots of baby mosquitoes, and so the population continues and expands.
[But if we can release engineered sterile males into the population, they will court and mate wild females, but have no viable progeny.]

7. But if we can release engineered sterile males into the population, they will court and mate wild females, but have no viable progeny. We call this system RIDL, so these are RIDL males. If we can release enough RIDL insects for long enough, the target population will decline and collapse.
This might sound crazy, but it has some big advantages. The sterile males will mate only with females of the same species, and not with anything else, so there are no off-target effects on other insects. And these males will seek out those females for you.
People often ask: can you rear enough RIDL males – after all there are a lot of mosquitoes out there.
RIDL®

8. But if we can release engineered sterile males into the population, they will court and mate wild females, but have no viable progeny. We call this system RIDL, so these are RIDL males. If we can release enough RIDL insects for long enough, the target population will decline and collapse.
This might sound crazy, but it has some big advantages. The sterile males will mate only with females of the same species, and not with anything else, so there are no off-target effects on other insects. And these males will seek out those females for you.
People often ask: can you rear enough RIDL males – after all there are a lot of mosquitoes out there.
RIDL

9. A Genetic Solution
Release engineered sterile males to prevent mosquito reproduction and so control dengue
“birth control for mosquitoes”
Sterile male mosquitoes actively seek females
Find mosquitoes better than human inspectors
Based on Sterile Insect Technique
How it works
Rear millions of insects
Sterilise with irradiation
Release over wide area
Sterile males mate with wild females: progeny don’t survive
Pest population declines
Species-specific
Used for over 50 years
WHO

10. New World Screwworm Eradicated
New World screwworm eliminated on a continental scale by a rolling programme of release of sterile insects. 10

11. RIDL: fail-safe / replacing radiation
RIDL insects are genetically sterile
Repressible
Release homozygous males X
Bi-sex lethal
Female-specific lethal X
Introgression of genes through male line

12. RIDL®: molecular biology
Controllable Gene Expression
Antidote (Tc)
Female death
death
tTA
effector
promoter
tetO
Female specificity
Thomas et al Science 287: Fu et al PNAS 107:

13. Act4-tTA + tetO-DsRed (DsRed) tTA effector OX3545F Act4 promoter
tetO + promoter
OX3545F
Flight muscles only

14. OX3604C RIDL mosquitoes Males Females
Flightless mosquitoes cannot survive in wild (or find hosts). Unable to mate even in laboratory. Males have normal flight ability, as have females given antidote as larvae.

15. fsRIDL phased trials – large lab cage trials
RIDL cage trials performed in Colorado State University (Megan Wise, Bill Black) showing suppression of target population

16. RIDL: fail-safe / replacing radiation
RIDL insects are genetically sterile
Repressible
Release homozygous males X
Bi-sex lethal
Female-specific lethal X
Introgression of genes through male line

17. Development trials First open release Grand Cayman 2009
10km
First open release Grand Cayman 2009
mating of RIDL males to local females
excellent mating competitiveness
provided data for suppression trial 2010
500m
Each Area approx 16 Ha (40 acres)
No conventional control for Aedes aegypti
Release period May-Oct 2010 with pre- and post-release monitoring

18. Cayman field trial 2010 Trial was complete success; all endpoints met
Cayman field trial 2010
Aedes aegypti populations show seasonal variation in Cayman, driven by rainfall. Control area – Area C, green line – shows this pattern (as did other control areas, omitted for clarity), illustrated by fitted polynomial trend line. Release area – Area A – shows clear suppression from beginning of August. Release period finished Oct 15. for last 7 weeks, release area had average ovitrap index of 10% vs control area 50% - an 80% reduction in the treated area relative to control. Since ovitrap index is somewhat non-linear, this probably represents suppression of the mosquito population by somewhat more than 80%. The degree of suppression is limited by immigration from adjacent areas, e.g. Area B, which was untreated and had a high mosquito population (resembling Area A).
Note that the control and treatment sites are close, so share environmental effects. The two plots therefore largely parallel each other in terms of week-to-week variation, with the additional effect of suppression in Area A.
Trial was complete success; all endpoints met
Clear suppression from early August
Sustained release of RIDL OX513A males can suppress a field population of Aedes aegypti mosquitoes
Maximum degree of suppression limited by immigration
GM mosquitoes can perform successfully in the field

19. Bringing new technology to the field
Technical
Community
Regulatory
RIDL
Nature

20. Regulatory Progress
The USDA completed (2008) an Environmental Impact Statement (EIS) on the use of autocidal technology (RIDL) in fruit flies and PBW
Record of Decision: this is the environmentally preferred alternative
North American Plant Protection Organization (NAPPO) standard signed late 2007
MosqGuide: WHO/TDR develop guidance for use of GM mosquitoes for disease control
Oxitec transgenic insect approvals
Multiple movement and contained trial approvals (Medfly, Mexfly, pink bollworm, Aedes aegypti, Ae. albopictus)
Open field release approvals in the USA (PBW 2006,7,8)
Released 15 million Oxitec pink bollworm from aircraft over 2500 acres (2008)

21. Community Engagement Focus Group: reactions to Oxitec approach
breakthrough!
good news
kills larvae
good – something worth trying going forward
provides immediate solution / result in preventing Dengue
seen as a long term solution cf other methods which are all seen as short term
some even welcome the research team to test the technique in their community
From TNS Malaysia
Community engagement
Malaysian Health Minister Datuk Seri Liow Tiong Lai “We see it as the most efficient and fastest way in eradicating Aedes mosquitoes from our local environment,” Liow said, adding that Aedes is not a species endemic [native] to Malaysia. Monday 11th Oct 2010

22. Frequently Asked Questions
Will the genetic modification spread outside the release area? The released mosquitoes and their progeny will die so this is a ‘self limiting’ approach, with no permanent change to the wild mosquito population. The large fitness cost (and no component advantage) also prevents spread. Do mosquitoes provide valuable ecosystem services (e.g. food chain, pollinators)? Aedes aegypti originated in Africa and only achieved pan-tropical distribution in the 1930s. Therefore in most countries it is not a native species. There are no birds, fish or other insects that feed exclusively on it and therefore reducing the number of Aedes aegypti is most unlikely to have negative impacts on the environment. If one mosquito suppressed, will a worse one replace (niche replacement)? Aedes aegypti it occupies an unusual, human-associated niche normally empty in its absence. Aedes albopictus, a potential alternative, is an inferior vector of dengue. RIDL strains are also available for Aedes albopictus. Can we release enough (feasibility, economics)? Long history of success and data from SIT implies ‘yes’, as do modelling and data so far.

23. HEALTHY PEOPLE HEALTHY ENVIRONMENT
Acknowledgements
Greg Simmons
Bob Staten (rtd)
Tom Miller (UCR)
Angi Harris
Bill Petrie
IMR, Malaysia
Lee Han Lim
PI: Tony James
PI: John Mumford 23

24. Thank you… 24