Theoretical Approaches to Using Genetic Manipulation to Control Disease Transmission

Michael Riehle University of Arizona Why do we need novel control strategies for mosquitoes? Current Control Strategies Parasite Control

Anopheles gambiae stephensi

Insecticide treated Indoor residual Antimalarial drugs Vaccine bednets spraying combination RTS,S/AS01 therapy (ACT) (39% efficacy) Challenges with current control strategies

Artemisinin Resistance

WHO World Malaria Report 2015 Pyrethroid Resistance

Ashley et al., N Engl J Med 2014; 371:411-423 Current Arboviral Control Strategies

Vector Control Pathogen Control

Aedes aegypti

No drug treatment Vaccine ULV spraying Larviciding Dengue-Dengvaxia® Challenges with current control strategies

RANSON, Hilary; BURHANI, Joseph; LUMJUAN, Nongkran and BLACK IV, William C. Insecticide resistance in dengue vectors. TropIKA.net [online]. 2010, vol.1, n.1 What are our other options???

1. Sterile technique (SIT) • Release of carrying a Dominant Lethal (RIDL)

2. Wolbochia infected mosquitoes

3. Population replacement strategies Sterile Insect Technique (SIT)

Dr. Edward Knipling's theorized that insect pests, such as the screwworm, could be controlled by inundating wild populations with the introduction of numerically overwhelming sterile males.

The egg masses laid by fertile females mated with sterile males would never hatch into the destructive larvae.

Reproduction would drop with each generation, eventually to reach zero if sterile flies continued to be introduced.

Known as the Sterile Insect Technique (SIT), Knipling focused on ways to use his method with the screwworm fly. Sterile Insect Technique (SIT) By the late 1930's, most elements for screwworm eradication were in place:

1. Knowledge of screwworm as a separate species with a dependence on live hosts

2. Bushland's technique for raising large numbers of the flies

3. Knipling's understanding of the mating patterns of the species

4. Knipling's theory for controlling the by the mass release of sterile males.

5. A fifth crucial element, a way to sterilize large numbers of screwworms, arrived with the assistance of the Oak Ridge National Laboratory and the loan of cobalt-60 gamma ray equipment Sterile Insect Technique (SIT) Sterile Insect Technique (SIT)

While this concept should apply to mosquitoes there are two main problems

Survival Release of and fitness potential disease vectors Release of Insects carrying a Dominant Lethal (RIDL) How does it work?

Wilke, André Barretto Bruno, et al. (2009) Release of Insects carrying a Dominant Lethal (RIDL)

Wilke, André Barretto Bruno, et al. "Mini-review: genetic enhancements to the sterile insect technique to control mosquito populations." AsPac J Mol Biol Biotechnol 17.3 (2009): 65-74. Release of Insects carrying a Dominant Lethal (RIDL) Pros and Cons

Pros Cons Minimal non-target effects Can’t release females Based on a proven technology Must manually sort males Males will seek out females Infrastructure for large scale breeding Allows for immature competition Concerns with releasing transgenic insect Reduces nuisance biting Leaves ecological void Self-limiting Self-limiting RIDL - Field examples

2009 Grand Cayman

Harris et al. Nat. Biotech. 30(9) 828-30

2012 Iteberaba Brazil

Nimmo - Sex specific RIDL – AKA RIDL 2.0 Sex specific RIDL – AKA RIDL 2.0

Non-transgenic Transgenic Sex specific RIDL – AKA RIDL 2.0 Pros and Cons

Pros Cons Minimal non-target effects Can’t release females Based on a proven technology Infrastructure for large scale breeding Males will seek out females Concerns with releasing transgenic insect Allows for immature competition Leaves ecological void Automatic sorting – female lethal Self-sustaining Reduces nuisance biting Self-sustaining

Has not been tested in the field Using bacteria to control dengue and Zika Wolbachia

Wolbachia is an intracellular bacterial symbiont found in a wide range of arthropods.

wMel infected ovaries

Credit: Simon Moule/Eliminate Dengue Using bacteria to control dengue and Zika How it works

Eliminate Dengue: Our Challenge Using bacteria to control dengue and Zika Lab Results

Walker, Thomas, et al. "The wMel Wolbachia strain blocks dengue and invades caged Dengue virus populations." Nature 476.7361 (2011): 450-453. Using bacteria to control dengue and Zika Lab Results

Aliota, Matthew T., et al. "The w Mel Strain of Wolbachia Reduces Transmission of Virus in Aedes aegypti." PLoS Negl Trop Dis 10.4 Chikungunya virus (2016): e0004677. Using bacteria to control dengue and Zika Lab Results

Aliota, Matthew T., et al. "The wMel strain of Wolbachia Reduces Transmission of Zika virus by Aedes aegypti." Scientific reports 6 (2016). Using bacteria to control dengue and Zika Field Results Using bacteria to control dengue and Zika Field Results

In 2011 two field releases were performed in

Sustained releases of wMel-infected mosquitoes over a 10-week period.

Five weeks following the last release, Wolbachia frequencies had reached almost 100 percent in wild populations at both locations.

Persisted over time.

Low level of uninfected Immigration? Using bacteria to control dengue and Zika Pros and Cons

Pros Cons Minimal non-target effects Mode of anti-viral action unknown Unique killing mechanism Resistance? Males will seek out females Studies on effectiveness against No ecological void arboviruses needed Self-sustaining with drive system Not recallable Does not affect nuisance biting Population replacement with genetically engineered mosquitoes Population replacement – how does it work?

Genetically engineer a mosquito that:

• Kills the pathogen in the mosquito

• Blocks the pathogen from developing within the mosquito

• Shortens the mosquito’s lifespan so that pathogens do not have time to complete their development How to engineer a malaria resistant mosquito

1. A method for genetically engineering mosquitoes

2. Anti-pathogen molecules

3. An effective genetic drive mechanism Transposable elements MEDEA CRISPR/Cas9 Components of a transformation system DNA delivery system: electroporation microinjection lipofection biolistics Components of a transformation system Active Class II elements:

P element: First identified in Drosophila Rubin and Spradling (1982) used the P element to restore color to Drosophila white eye mutants Researches spent ~10 years trying to get the P element to work in other insects Only works in Drosophila Components of a transformation system Active Class II elements:

~Size Length of Target-site Element Family Origin (kb) ITRs specificity Hermes hAT Musca 2.7 17 GTNCAGAC domestica MosI Tc1- Drosophila 1.3 ~30 TA mariner mariner mauritiana Minos Tc1- Drosophila 1.4 254 TA mariner hydei piggyBac novel Lepidopteran 2.5 13 TTAA baculovirus Components of a transformation system Marker genes:

Physiological (select) antibiotic resistance genes (neomycin, hygromicin) insecticide resistance (OPD, dieldrin) Components of a transformation system Marker genes: Visible (screen) fluorescent proteins eye color Components of a transformation system Marker genes: Fluorescent markers predominate Greatest reason multiple markers can be used Components of a transformation system Promoters:

Actin 3XP3 Testes specific

Carboxypeptidase = Midgut specific Vitellogenin = Fat body specific Transformation procedures involve donor and helper constructs

Mos 1 mariner donor plasmid

MLH SV40 EGFP ACT 5C VG MAL-I N2 SV40 MRH

Marker gene Effector gene

Plasmid backbone

Mos 1 mariner helper plasmid

HSP82 promoter Mos 1 transposase

Plasmid backbone Transformation Procedures

• Donor and helper plasmids are injected into insect embryos (G0 generation) before formation of nuclear membranes (~2 h).

• Embryos are heat-shocked to induce transposase (often not necessary)

• Embryos are reared to adult stages (transient expression)

• Adults are mated to non- transgenic animals

• Progeny (G1) are scored for presence of marker genes and families established Microinjection of DNA into newly laid An. Stephensi eggs Other transformation systems Gene targeting systems Advantages: Known insertion site Reduced mortality Increased fitness Improved transformation efficiency

Disadvantages: More complex Additional marker is required Additional lines maintained Cre/loxP phiC31 Other transformation systems CRSPR/Cas9

Advantages: Site directed insertion No docking strains required Efficient knock-out Inexpensive

Disadvantages: Low efficiency for knock-ins

New England Biolabs How to kill the pathogen Effector Molecules

Malaria Parasites Arboviruses Antimicrobial peptides Antimicrobial peptides Single chain antibodies RNAi constructs Reactive Oxygen Species RNAi Pathway RNAi constructs Promoters/lethal genes How to kill the pathogen Drive Systems

Mendelian inheritance is not going to be sufficient Too many mosquitoes need to be released Fitness costs

Thus we need a genetic drive system that can induce inheritance of the anti-pathogen gene at an accelerated rate.

These can include: Transposable elements Wolbachia MEDEA CRISPR/Cas9 Maternal-Effect Dominant Embryonic Arrest MEDEA Crispr/Cas9 Drive

Gantz et al. PNAS 2015 112 (49) E6736-E6743 Other concerns

Logistics:

Can you rear enough mosquitoes?

Can you transport the mosquitoes to areas of need? Poor/no roads Political unrest Lack of public cooperation Other concerns

Ethics:

You are releasing a flying transgenic vector

Does not respect international boundaries

Will require the combined support of governments, health organizations, philanthropy and the general public Other concerns

Multiple mosquito subspecies:

For example - The most important malaria vector is the Anopheles gambiae complex

Consists of at least seven species

Many reproductively isolated subspecies Where are we at?

RIDL – Multiple field releases with a proven reduction in Ae. aegypti populations wMel – Multiple field releases with persistant wolbachia establishment. Antiviral effectiveness must still be proven

Population replacement – Ability to engineer pathogen resistant mosquitoes. However, no drive mechanism and numerous ethical and logistical issues. Acknowledgements

Jenya Koch Vanessa Corby-Harris

UC-Davis Group U. Georgia Group Support Dr. Shirley Luckhart Dr. Mark Brown Dr. Nazzy Pakpour Dr. Andrew Nuss Dr. Cecilia Giulivi Dr. Bo Wang Kong Wai Cheung Eric Hauck Jose Pietri Anna Drexler