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My name is Anthony James. I'm a split appointment in the departments of microbiology and molecular biology and then molecular biology and biochemistry here at the University of California Irvine. My laboratory works to develop answers to the simple question of whether or not genetics has anything to contribute to the control of vector-borne diseases.

And our particular case, we're interested in mosquito-borne diseases, malaria and dengue fever. Malaria is the largest vector-borne disease in terms of human mortality that kills one person about every 20 to 30 seconds, averaged over a year. Dengue virus isn't quite as significant in terms of human mortality, but is a major public health problem affecting nearly half the world's, potentially affecting nearly half the world's population with some 50, 000 a hundred thousand cases a year.

And, and in the tropical areas, current approaches for controlling Vector-borne diseases, specifically malaria and dengue viruses are focused on c control of the vector through the use of insecticides or reduction of larval habitat, larval and reproductive habitats. The the case of malaria, the use of therapeutic and prophylactic drugs and control measures that prevent access of people to mosquitoes. That includes the use of bed nets and other personal protective measures that, that prevent the mosquitoes from gaining access to the people.

So we ask the question as I indicated whether or not genetics had anything to offer, and there are two areas in which we we're considering genetics. One is to develop genetic techniques that reduce populations of mosquitoes. This has been called population reduction.

And the second area is population replacement. And here the idea is to take genes that would confer resistance to specific pathogens and somehow get them to be into populations at high frequencies. We call this population replacement.

So there were two Interesting observations that let us think that genetics had a role to play in the control of vector-borne diseases. The first is if you look at all the insects or arthropods, and that includes ticks that feed on blood, you discover that not every blood feeding arthropod transmits every disease. So that suggests to you that there are some host restrictions of these pathogens, and that turns out to be true.

And when you think about host restrictions, you start thinking genetics. The second observation was that if you look at a population of insects that does transmit through very simple techniques, you can select within that group of insects a population which is highly susceptible and one which is highly refractory. Having done that, you can do simple genetic crosses and answer questions about how many genes are involved, whether or not there's a simple dominance, recessive relationships.

And from these observations, we, we had a very clear indication that genetics was important for a particular mosquito species to support the development of a specific pathogen. And the idea was, well, wouldn't it be great if we could somehow have those genes that confer resistance spread throughout all populations? And the advantages of this would be that we'd be targeting specifically the pathogen and in question and avoid use of things like insecticides, which have a lot of impact on non-target organisms or environmental management to reduce breeding sites, which have a profound, can have a profound effect on, on the environment.

And unlike the use of insecticides and prophylactic drugs, we don't expect it properly develop that these mosquitoes would ever, or the parasites that are raised in the mosquitoes would ever have any kind of a resistance to them. So the idea is to be able to develop a strategy where you have a completely disease resistant mosquito and Not have to worry about non-target effects. So when we looked At natural recurring raciness, the, the attractive approach was to see how if we could get some of these natural genes and increase their frequencies in populations, we could then have a, a, a disease resistant population.

But it turned out, at least in the early days, getting our hands physically on these genes was very difficult. So our group and a number of others took a totally synthetic approach where the idea was to build essentially a gene that could confer refractoriness. And under these circumstances, we divided the gene into two simple components, a control portion that would tell you when and where and how much of a particular substance to make.

And then an infect effector portion, which is the actual product that was made by that gene that could confer resistance. And so in our analysis of, of how we're gonna build these genes, we decided that it would be important and wise if the control portions would actually place the effector molecule in that part of the mosquito where the parasites actually were. And so we were looking for developmentally regulated genes that would allow us to put something in the right place.

And that something, once again is the effector molecule, and that's the actual portion of the molecule made by the gene that interferes directly with either the malaria parasites or the viruses. And for the malaria work, we developed what are called single chain antibodies that interfere with the bind to parasites and interfere with their ability to invade mosquito tissues. And for the dengue viruses, we develop what's called an RNAi strategy, which uses the cellular properties of a, of the mosquito to, to break down the viruses as they Attempt to replicate in the mosquito.

So when we first considered making These synthetic genes, we, we broke down the problem into component parts. And the first component part was to demonstrate in the laboratory that we could actually make a mosquito that normally would transmit a pathogen into one that could not. And, and in order to do that, as I was alluding to before, we needed to have control sequences that would express the effector molecule we would need to be able to make an effector molecule, and then we'd actually need some technology, transgenesis technology that would allow us to put this artificial gene into the mosquito.

And so for the past decade or so, that's what we've worked on and myself very much interested in, our colleagues interested and have, have made quite a few advances in that area. However, having developed these mosquitoes in the laboratory, we needed a mechanism to move that gene out of the laboratory into the field. And we needed some process that would allow this gene to increase at a frequency in the population that was, that would go beyond the types of increases that you would see with natural selection.

And so these types of mechanisms are called gene drive systems, and what they do is they select for insects that carry your gene of interest, and they have a number of properties, but the two major properties are that they are involved in or something that does occur as a result of mating, but increases a gene frequency at a rate much higher than you would expect through natural selection for some Selective advantage on that gene. So when we thought about developing these Gene drive systems, we recognized immediately that we're talking about introduction of genetically modified organisms into the open environment. And not too surprisingly, there were significant concerns and anxieties associated with redu, with releasing genetically engineered materials into the wild.

And it was important for us to identify what significant concerns would be associated with the use of genetically modified organisms, and then couple them with the science that we have in an effort to reduce the, the, the issues associated with using them. And so we developed what we call design criteria that take into account in addition to the biological nature of the, of the, of the organisms we're developing in laboratory criteria, but also criteria that associated with, with sociological and cultural and other concerns that people would have. So these become the design criteria.

So three of em that I'm gonna talk about briefly here, overlap in, in these areas. The first is that whatever we develop in terms of a gene drive system has to work in what's you would call a human timeframe, not an evolutionary timeframe. So this is something that has to spread a gene quickly enough that we can actually see something occur as a consequence of it.

And we're thinking on the order of say, five to 10 years. And this was an idea first developed by Nora pki who recognized that, you know, given 10, 000 years we could do something, but the gene drive system that we're talking about had to work within a much more human timeframe. The second and probably largest concern and criteria that we have is that whatever we develop work on the target organism only that is, it doesn't have the ability or is disabled if it, if it actually gets outside of our target insect.

And this is important because most people's concerns are that you make something in the laboratory and they think that's fine, but that it'll get out into nature and somehow spread to non-target target species. And so one of our major design concerns is that we build something that would only function in the target species and be disabled if somehow crossed out of, out of the target species. The third thing that's fairly significant, of course, is that, that it has to work.

Working means more than just the science and the restriction to species, for example. It has to be cost effective. It has to be something which actually works in a reasonable framework, and so that it's something that can be affordable to do.

So those are a number of the design criteria that we, we've looked at. They're not all of em. And I, and I imagine that some of the criteria will change as our experience goes along, but I Imagine those three that will, will stay the same.

So before this whole Idea actually, and and practices can be implemented, a number of things have to happen. Getting back to this idea that, you know, first of all, it has to work. We need to be able to show in the laboratory that the insects that we develop truly are resistant to the pathogens that we're targeting.

And then we have to be able to show that the gene drive mechanism functions the way we expect to do that. In the latter case, we are planning to do cage trials, contain cage trials so there's no release into the field that will put the gene into an endogenous mosquito or derived from the endogenous mosquitoes in the target site, and then ask, does it have the ability to move? So this is going to be important for us to do.

However, I think it's critical to realize that it's not a scientist like myself who's gonna do the recommending that a field trial ever be implemented. This will actually be the result of mathematical modelers who using the parameters that they develop and, and that we provide can give a public health institution a strong idea of whether or not what we've developed is actually gonna work in a cost effective manner. So the, the recommendations will actually come from public health professionals who are, have expertise in modeling, and they're the ones that are gonna be able to say something's a no or a go or a no go.

We don't anticipate that the laboratory people like myself will have much input beyond providing the, the, the raw data To the modelers in order to, for them to use that. When we first started doing this work, We looked around when we say we, cause my colleagues and I are work as a team in this, but we looked around for preexisting structures that would allow us to evaluate what we would need to do in terms of addressing the ethical situation of using genetically modified organisms. And while there are quite a bit of data available for agricultural uses and, and the use of of, of plants, et cetera, there was very little in the published literature about the use of genetically modified insects.

And so as part of our major project, we had to include a component that dealt with the ethical, social and legal aspects of, of using genetically engineered mosquitoes. And as a consequence of that, it's been an amazing learning curve in terms of bringing every member of the group up to speed on what's required to actually to make this happen. And let me give you a few examples.

Most countries in the world who Have these major diseases do not have regulatory structures in place that can pass critical judgments on the use of genetically modified organisms. And so it became extremely important for us to identify countries that had a proper regulatory infrastructure with whom we could, we could consult and, and interact. And the history of a situation like this is that countries often were confronted with the introduction of a genetically modified organism once again, mostly in agricultural agriculturally derived product, say genetically modified corn or papayas or rice.

And the country lacking the regulatory structure to deal with it would quickly pass a law that that would prevent the introduction of any genetically modified organisms at all. If you think about it in a political sense, this is a smart thing to do. That is if you, if you're not prepared to deal with it, shut it down and, and give yourself a chance to develop the pro, the necessary structure to, to analyze it and decide whether or not there's a, a problem here.

And once this happens, countries put into place the proper regulatory structure and then they reverse the laws and start letting whatever the genetically modified organism is, at least come under review and perhaps ultimately be used. So in thinking about our work, we couldn't pick a country where we were gonna be the precipitating event for them to develop their, their, the regulatory infrastructure. We had to find countries that had already been through this process and already had a robust regulatory system in place.

The reason we wanted to do that is we wouldn't have the time actually to, to be once again the precipitating event. Having said that, then we wanted to be also in a country where there was a very well vertically integrated public health system, meaning that we would be addressing concerns both at the local level and then on up through the, the larger municipal county state or, and then federal equivalent in, in, in the regulatory structure. And we wanted to make sure that decisions that were made that, that, to go ahead, for example, were were validated at every level.

It wasn't something that the central government or federal government was gonna pose on a local government. We wanted it to be something that, that everybody agreed to. So we had to actually come into a circumstance where there was a really well integrated vertical public health system.

And then we're addressing issues, for example, of what it means to have community Consent. How do you define a community? We define the community at this point is All the people who, who are or potentially could be impacted in the immediate sense by the work that we're conducting.

But that turn, turn turns out not to be trivial, to define and under circumstances where you are going after community consent, what does it mean to actually have community consent? Is one negative vote enough to shut it down? What, you know, these are things that have to be worked out.

So these, these are Some of the ethical issues that we're confronting. One of our major Efforts in this work though, is to make sure that from the beginning we have full participation of the people in the field site, in, in our deliberations, in, in our efforts and to this, to make this happen. For example, we have a website that's really accessible to everybody and we have full participation of our prospective labor laboratories collaborating laboratories with people at the field site so that they're part of the planning process And the implementation process.