Nanotechnology, Medicine & Aerosystem Genetically engineered (GE) Aedes aegypti Mosquito

Genetically engineered (GE) male Aedes aegypti of the OX5034 strain for use in mosquito control. Aedes aegypti is a known vector for human diseases associated with Zika, dengue, and chikungunya viruses. Oxitec’s novel approach to mosquito control uses the release of male OX5034 mosquitoes carrying a “female-specific self-limiting gene” to mate with wild females. When male OX5034 Aedes aegypti homozygous for the self-limiting gene (carrying two copies of the gene) are released into the environment and mate with wild Aedes aegypti females, their offspring inherit a single copy of the self-limiting gene (so are hemizygous). The self-limiting gene kills only female offspring (carrying one copy of the self-limiting gene), which die at early larval stages of development, while hemizygous males will survive to pass the OX5034 genes on to subsequent generations. Laboratory tests show that 100% of the resulting female offspring will die before reaching adulthood. Hence the OX5034 mosquito can be considered a sex- and species-specific larvicide targeting only female Aedes aegypti.

Introduction

Expression of tTAV-OX5034 is regulated by tetracycline or one of its analogues. Tetracyclines bind to tTAV protein, preventing it from activating transcription. Thus, when either tetracycline or one of its analogues is absent from the OX5034 mosquito larval diet, tTAV-OX5034 protein causes lethality in females carrying at least one copy of the construct, including the progeny of mating between OX5034 homozygous males and wild Ae. aegypti females. Tetracycline Trans-Activator Variant (tTAV-OX5034) protein and the genetic material (from vector pOX5034) necessary to produce the protein in vivo

Figure: Schematic representation of the OX5034 female-lethal trait mediated through sex-specific tTAV-OX5034 expression. Top: In the absence of tetracyclines, basal expression of tTAV-OX5034 in female OX5034 Ae. Aegypti results in the production of the tTAV-OX5034 protein. The Aeadsx splicing module located at the 5’-end of the tTAV-OX5034 gene is alternately spliced in males and females, leading to the preferential expression of the fulllength tTAV-OX5034 mRNA isoform in females. Once the protein is translated it is cleaved by endogenous deubiquitinases at the UBQ-tTAV junction, releasing tTAV. tTAV then dimerizes (purple ovals). The positive feedback loop is closed when the VP16-domains of the tTAV protein bind to the tetO7 operator, which enhances the expression of the tTAV-OX5034 gene. Through this mechanism of overexpression, cellular functions are affected resulting in cell death in the developing larvae. Bottom: In the absence of tetracyclines, basal expression of tTAVOX5034 still occurs. Because tTAV preferentially binds to tetracycline than to tetO7, expression is not enhanced.

OX5034 is described as a species-specific female larvicide, or “male-selecting” larvicide, that results in all-male progeny in the absence of tetracycline in the larval diet due to a female-specific self-limiting gene. With continued field releases of OX5034 homozygous males, the. Ae. aegypti population is expected to progressively decline due to the reduced number of females emerging in the area. Specifically, when OX5034 homozygous males are released into the environment and mate with wild Ae. aegypti females, their offspring inherit a single copy of the self-limiting gene. The self-limiting gene kills only female offspring while hemizygous males survive to pass on the OX5034 self-limiting gene further. As the self-limiting gene is inherited in a Mendelian fashion, half of the offspring resulting from a mating between an OX5034 hemizygous male and a wild female would not inherit the self-limiting gene but would still inherit OX5034 strain genetics. This results in both male and female mosquitoes with OX5034 strain genetics.

Additionally, a recent paper examining the applicant’s 1st generation product, OX513A, found evidence of introgression after releases in Brazil of males containing the self-limiting tTAV gene (Evans et al. 2019). Although that paper investigated OX513A mosquitoes, the findings are relevant to the evaluation of OX5034 because the degree of introgression is likely to be significantly higher than that of the OX513A strain due to higher larval survival rates (approx. 5% in OX513A versus 50% in OX5034). Therefore, given that introgression of OX5034 strain genetics will occur during releases, it is pertinent to examine potential associated risks for humans and the environment. Traits associated with a disease vectoring species such as Ae. aegypti that may carry risk if introgressed into a wild population are likely to be linked to vectorial capacity, including vector competence, fecundity, and longevity.

The U.S. Food & Drug Administration regulates mosquito-related products that are intended to reduce the viral/pathogen load within a mosquito or are intended to prevent mosquito-borne disease in humans or animals (USFDA, 2017). EPA, on the other hand, regulates products intended to reduce the population of mosquitoes as pesticides, such as OX5034. Under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA), EPA evaluates pesticide products to ensure that the products do not cause unreasonable adverse effects on humans or the environment. Consistent with the FIFRA standard, EPA is reviewing several factors that could contribute to the ability of the OX5034 mosquito to serve as a disease vector from the perspective that an increased ability to vector disease could result in a risk to humans or the environment.

Vectorial capacity is influenced by a number of traits impacted by gene-environment interactions and is confounded by both intrinsic and extrinsic variables. The applicant stated that during releases of OX5034 mosquitoes, the local population is expected to decline and therefore should have a reduced vectorial capacity due to decreased population density.

Environmental Protection Agency’s (EPA) decision to grant Oxitec an Experimental Use Permit (EUP) for piloting its 2nd generation Friendly Aedes aegypti mosquito technology, the result of an in-depth and rigorous scientific review process that included technical support from the U.S. Centers for Disease Control (CDC) and an expansive assessment of the technology and its safety relating to humans and the environment.

The EPA’s media statement below:

Ø. Oxitec to carry out demonstration projects with its safe, environmentally sustainable Aedes aegypti technology designed to control the mosquito that transmits dengue, Zika, chikungunya and yellow fever.

Ø. Approval adds to U.S. Food and Drug Administration’s (FDA)’s ‘Finding of No Significant Impact’ on human, animals or the environment for Oxitec’s mosquito technology (2016) and U.S. Department of Agriculture’s (USDA) approvals of field trials of two Oxitec agricultural pests.

Ø. U.S. Centres for Disease Control (CDC) supported EPA’s scientific review of application.

. Successful deployments of Oxitec insect technologies worldwide, and driven by a team of experts from the US, UK, Brazil, and a dozen other countries, Oxitec’s new Aedes aegypti technology represents a significant leap forward in efforts to provide municipalities and communities throughout the US with new, safe, sustainable and scalable tools to combat the growing threat posed by the invasive Aedes aegypti mosquito, which transmits devastating diseases such as dengue, Zika and chikungunya.

Oxitec has already completed successful deployments of this new Aedes aegypti technology in Brazil, demonstrating its ability to quickly and significantly suppress wild Aedes aegypti using easy-to-use, just-add-water deployment devices, thereby eliminating the need for costly production facilities and labor-intensive adult mosquito releases required by other insect-based technologies. This technology is designed to give governments and communities a powerful new solution that can scale effectively without the need for complex infrastructure and serve as a stand-alone solution or as a valuable component of integrated vector management programs.

EPA APPROVES EXPERIMENTAL USE PERMIT TO TEST INNOVATIVE BIOPESTICIDE TOOL TO BETTER PROTECT PUBLIC HEALTH

Environmental Protection Agency (EPA) has granted an experimental use permit (EUP) to Oxitec Ltd. to field test the use of genetically engineered Aedes aegypti mosquitoes as a way to reduce mosquito populations to protect public health from mosquito-borne illnesses.

During these field tests, Oxitec will release into the environment male mosquitoes genetically modified to carry a protein that will inhibit the survival of their female offspring when they mate with wild female mosquitoes. The male offspring will survive to become fully functional adults with the same genetic modification, providing multi-generational effectiveness that could ultimately lead to a reduction in Aedes aegypti mosquito populations in the release areas. EPA anticipates that this could be an effective tool to combat the spread of certain mosquito-borne diseases like the Zika virus in light of growing resistance to current insecticides.

. Male mosquitoes will be released into the environment and they do not bite people, they will not pose a risk to people. It is also anticipated that there would be no adverse effects to animals such as bats and fish in the environment.

• EPA has also maintained the right to cancel the EUP at any point during the 24-month period if unforeseen outcomes occur.

Similarities and differences between OX5034 and OX513A

Aedes aegypti mosquitoes Oxitec’s l “ generation self-limiting mosquito technology (OX513A), successfully deployed in multiple locations including in Brazil, the Cayman Islands and Panama, has been succeeded by the new 2nd generation self-limiting mosquito, OX5034. The OX5034 mosquito carries many of the key features of OX513A that made it a safe, effective control method for reducing Aedes aegypti mosquito populations. These include effective mosquito control, non-toxic and non-allergenic active and inert ingredients, a lack of direct effects on non-targeted species, and no long-term effects or chemical residues in the environment. OX5034 also has several additional features, including genetic sex-separation, which enables more cost-effective production and release of only male mosquitoes, and a brighter fluorescent marker, which enables field monitoring in all life stages of the mosquito. The key similarities and differences between OX513A and OX5034 are highlighted in the table below.

In Conclusion

The DNA sequence of tTAV -OX5034 contains additional features (relative to OX513A) that enable its expression only in female mosquitoes, but the protein sequence is the same (99.4 % identity). The DNA sequence of DsRed2- OX5034 contains additional features that enable brighter expression in all mosquito life stages, but the fluorescent protein domain is the same as in OX513A (98.6% identity). All have the same non-toxic and non-allergenic safety profile.

Reference:

. EPA-HQ-OPP-2019–0274

. Beerntsen, B. T., A. A. James, and B. M. Christensen. 2000. Genetics of mosquito vector competence. Microbiology and Molecular Biology Reviews 64:115–137.