Patricia F. Fitzpatrick Dimond Ph.D. Technical Editor of Clinical OMICs President of BioInsight Communications

Genetically modified mosquitos could be a way to curb the spread of dengue fever in Florida.

In 2010, a dengue outbreak was reported in the Florida Keys, in Florida’s Monroe County in southern Florida, marking the first outbreak of the disease in seventy-three years—an event hardly conducive to tourism.

Dengue virus (DENV) causes a severe flu-like illness that is transmitted to humans through the bite of mosquitoes, specifically Aedes aegypti, the principal vector of the disease. Also known as breakbone fever, four different viruses cause dengue, greatly complicating potential development of an anti-viral vaccine.

Dengue symptoms include chills, high fever, vomiting, and rashes, with the most distinctive accompanying symptom being agonizing pain behind the eyes and in arm and leg bones. Once the the fever subsides, lack of appetite and deep exhaustion may leave affected individuals unable to work for weeks or months. Dengue can vary from mild to severe; the more severe forms include dengue shock syndrome and dengue hemorrhagic fever (DHF). Patients who develop the more serious forms of dengue fever usually require hospitalization.

Mosquito eradication programs initiated at the end of World War II broke the chain of transmission between humans and insects, and by the end of the war, dengue had become a tropical illness that no longer occurred in the U.S. in significant outbreaks.

But dengue is back with a vengeance in the U.S., and apparently never left, as indicated by the recent Florida outbreak, and has remained an endemic disease in Puerto Rico. Periodic dengue activity has occurred since since 1963, with three to nine thousand suspected dengue cases reported during nonoutbreak years.





Disease Control

Control of the disease in Puerto Rico from 1970–1980s relied on pesticide use and household inspections, approaches that the CDC has described as “unsustainable.” The focus shifted toward citizen involvement in the control of vector “breeding sites” in yards and homes, personal protection through use of insect repellent, and education on recognition of symptoms with the need to seek timely medical care.

Dengue remains a major human plague in other parts of the world. According to the NY Times, 30,002 people in India had been sickened with dengue fever through October in 2012, a 59% jump from the 18,860 recorded for all of 2011 with the real number of Indians who get dengue fever annually closer to millions. “I’d conservatively estimate that there are 37 million dengue infections occurring every year in India, and maybe 227,500 hospitalizations,” said Scott Halstead, M.D., a tropical disease expert focused on dengue research.

Currently there is no vaccine, or a specific treatment for dengue, leaving management of its vector, the Aedes aegypti mosquito, the only option for controlling the disease. And the mosquitoes have, some reports say, developed resistance to insecticides use to control them.





Genetically Modified Mosquitoes

Given great concern in the Florida Keys about the region’s fragile ecosystem, mosquito control officials in the Keys have been searching for alternatives to pesticide use, which can and have negatively impacted public health and wildlife. Local mosquito control officials in the Florida Keys are awaiting approval from the federal government to begin releasing hundreds of thousands of genetically modified mosquitoes developed by British company Oxitec to stop the spread of the disease.

Oxitec is in discussions with the FDA regarding potential release its RIDL® (Release of Insects carrying a Dominant Lethal) male mosquitoes in the Keys. As Oxitec explains, by “sterile” it means that although the males do produce sperm and can fertilize the female’s eggs. But their offspring are “inviable”, meaning that they die at a very early stage of development due to the lethal gene construct passed on by the altered males.

The sterile OX513A mosquitoes were produced by introducing, via a piggyBac helper plasmid injected in mosquito eggs, the OX513 transposon. The OX513 construct encodes the tetracycline-repressible transcription activator (tTA). Once the mosquitoes were developed with the stably-integrated construct, the company was able to breed them, and says it has been breeding them now for over 100 generations.

When expressed at high levels the tTA protein proves lethal, probably due to transcriptional “squelching” and/or interference with ubiquitin-dependent proteolysis.

In a deadly genetic positive feedback cycle, when expressed in LA513 the tTA protein can keep its own expression turned on by binding to an upstream operator sequence, thus driving expression of tTA from a nearby minimal promoter, which in turn binds to the activator tetO, creating a positive feedback system.

But tetracycline, which binds tTA, prevents the activator from interacting with tetO, allowing batches of transgenic mosquitoes to be grown in the presence of the antibiotic. The resultant transgenic Aedes eggs are collected for hatching at a trial site, and the smaller male pupae are sorted from females and on maturity released into the field, where breeding with wild-type female mosquitoes results in sterile mating.

Oxitec scientists have conducted two series of field tests, one in the Cayman Islands and one in Brazil, to determine whether the altered mosquitoes could survive in the wild, their ability to compete for female attention with wild-type males, and whether they could successfully pass on the repressible lethal genetic system that would ensure their offspring would not survive.

Oxitec founder and chief scientific officer Luke Alphey, Ph.D., and the developer of the RIDL system, told GEN, “The trials turned out as exactly as we hoped with all endpoints met. The first trial asked the question whether these mosquitoes could successfully survive in the wild and mate with females in competition with wild-type males. That is fundamental to any kind of sterile insect method. The sterile mosquitoes survived and mated and we could then calculate how many males we would need to release over a specific time period in order to suppress the wild population.”

The key end point of second trial, he said, was “to determine whether we could suppress the population of wild type mosquitoes, and we saw an 80% suppression in the population.”





Regulatory Mosquito Netting

From a regulatory standpoint, Dr. Alphey said, “We are in discussions with the FDA and that’s a good thing because it took a while to find a regulatory agency that would pick up the ball. The first release of a GM insect anywhere in the world was in the U.S. in Arizona, and that was for a plant pest. So it wasn’t immediately clear who in the regulatory world was in charge because mosquitoes are not plant pests, so a different regulatory path was needed.” And, he noted, “Credibility through regulation is important for us to reassure everybody that release of these mosquitoes is safe.”

The company ultimately entered into discussions with the FDA after the Centers for Disease Control and Prevention, Department of Agriculture, U.S. Fish & Wildlife Service, Environmental Protection Agency, and other agencies, when approached as potential permit-givers, said they have no jurisdiction. The FDA themselves were initially among those organizations.

Dr. Alphey told GEN that “we have had very strong support among local populations” in the Caymans and in Brazil, because “people recognize the problem and that we don’t have other tools to deal with disease.”

But not so unanimous in Florida. Locals have doubts about the safety of the mosquito release, including people who believe news of the release will destroy the tourist industry, although the prospect of contracting dengue would certainly give potential visitors pause.

In April, the Key West City Commission approved a nonbinding resolution “opposing the introduction of genetically altered mosquitoes until further research is provided and approval is obtained from the applicable regulatory body.”

While awaiting a regulatory decision, Florida Keys Mosquito Control District executive director Michael Doyle plans on reassuring concerns of locals who remain opposed to the idea. A recent survey among Keys’ residents found that 30% supported it, 30% were against it, 8% were unaware, and the remainder were undecided. But Doyle says even if the majority turns against the project, he will proceed with it.

“My job is to protect them,” he says, “even if sometimes they don’t want to be protected.”

And with no other options to control the disease and no anti-dengue vaccine or adequate treatment to prevent its more lethal manifestations, local objections may have to yield to a recombinant mosquito alternative.

























































































































































Patricia Fitzpatrick Dimond, Ph.D. (drpdimond@comcast.net), is a principal at BioInsight Consulting.