Self-limiting Gene Research Articles

Elimination of a closed population of the yellow fever mosquito, Aedes aegypti, through releases of self-limiting male mosquitoes.

Establishment of novel mosquito control technologies such as the use of genetically engineered insects typically involves phased testing to generate robust data-sets that support its safe and effective use as a vector control tool. In this study, we demonstrate the ability of the transgenic self-limiting OX513A Aedes aegypti strain to suppress a wild type Ae. aegypti population in an outdoor containment facility in India. OX513A is a genetically engineered Ae. aegypti strain with a repressible dominant self-limiting gene. When male adult OX513A mate with wild female adults, a single copy of the self-limiting gene is inherited by all the progeny, leading to death of >95% of progeny during larval/pupal development. A wild-type population of Ae. aegypti was established and stabilized during a 14 week period in five paired field cage units, each consisting of control and treatment cages, followed by weekly releases of OX513A male adults to suppress the target population. The successive introductions of OX513A male adults led to a consistent decline in wild type numbers eventually resulting in the elimination of Ae. aegypti from all treated cages within 10 to 15 weeks of release. This study demonstrates that Ae. aegypti elimination may be a realistic and achievable target in relatively isolated environments.

Novel combination of CRISPR-based gene drives eliminates resistance and localises spread

Invasive species are among the major driving forces behind biodiversity loss. Gene drive technology may offer a humane, efficient and cost-effective method of control. For safe and effective deployment it is vital that a gene drive is both self-limiting and can overcome evolutionary resistance. We present HD-ClvR in this modelling study, a novel combination of CRISPR-based gene drives that eliminates resistance and localises spread. As a case study, we model HD-ClvR in the grey squirrel (Sciurus carolinensis), which is an invasive pest in the UK and responsible for both biodiversity and economic losses. HD-ClvR combats resistance allele formation by combining a homing gene drive with a cleave-and-rescue gene drive. The inclusion of a self-limiting daisyfield gene drive allows for controllable localisation based on animal supplementation. We use both randomly mating and spatial models to simulate this strategy. Our findings show that HD-ClvR could effectively control a targeted grey squirrel population, with little risk to other populations. HD-ClvR offers an efficient, self-limiting and controllable gene drive for managing invasive pests.

Development and testing of a novel killer-rescue self-limiting gene drive system in Drosophila melanogaster.

Here we report the development and testing of a novel self-limiting gene drive system, Killer-Rescue (K-R), in Drosophila melanogaster. This system is composed of an autoregulated Gal4 Killer (K) and a Gal4-activated Gal80 Rescue (R). Overexpression of Gal4 is lethal, but in the presence of R activation of Gal80 leads to much lower levels of Gal4 and rescue of lethality. We demonstrate that with a single 2 : 1 engineered to wild-type release, K drives R through the population and after nine generations, more than 98% of the population carry R and less than 2% of the population are wild-type flies. We discuss how this simple K-R gene drive system may be readily adapted for population replacement in a human health pest, Aedes aegypti, or for population suppression in an agricultural pest, Drosophila suzukii.

Invasion and migration of spatially self-limiting gene drives: A comparative analysis.

Recent advances in research on gene drives have produced genetic constructs that could theoretically spread a desired gene (payload) into all populations of a species, with a single release in one place. This attribute has advantages, but also comes with risks and ethical concerns. There has been a call for research on gene drive systems that are spatially and/or temporally self‐limiting. Here, we use a population genetics model to compare the expected characteristics of three spatially self‐limiting gene drive systems: one‐locus underdominance, two‐locus underdominance and daisy‐chain drives. We find large differences between these gene drives in the minimum release size required for successfully driving a payload into a population. The daisy‐chain system is the most efficient, requiring the smallest release, followed by the two‐locus underdominance system, and then the one‐locus underdominance system. However, when the target population exchanges migrants with a nontarget population, the gene drives requiring smaller releases suffer from higher risks of unintended spread. For payloads that incur relatively low fitness costs (up to 30%), a simple daisy‐chain drive is practically incapable of remaining localized, even with migration rates as low as 0.5% per generation. The two‐locus underdominance system can achieve localized spread under a broader range of migration rates and of payload fitness costs, while the one‐locus underdominance system largely remains localized. We also find differences in the extent of population alteration and in the permanence of the alteration achieved by the three gene drives. The two‐locus underdominance system does not always spread the payload to fixation, even after successful drive, while the daisy‐chain system can, for a small set of parameter values, achieve a temporally limited spread of the payload. These differences could affect the suitability of each gene drive for specific applications.

Self-Limiting OX513A Aedes aegypti Demonstrate Full Susceptibility to Currently Used Insecticidal Chemistries as Compared to Indian Wild-Type Aedes aegypti

OX513A Aedes aegypti is a genetically engineered strain carrying a self-limiting gene. Studies in several countries have shown the effectiveness of the strain at reducing pest Aedes aegypti populations. As a component of biosafety assessments relevant to Indian environments, OX513A and two Indian wild-type Ae. aegypti strains (from Aurangabad and Delhi) were tested for susceptibility to a range of commonly used insecticides in India, such as dichlorodiphenyltrichloroethane (DDT), malathion, deltamethrin, and permethrin using World Health Organization (WHO) testing kits and following WHO standard test procedures. Knockdown times (KDT) for all compounds were determined separately for male and female adults of the three mosquito strains. Results indicated that adults of OX513A, Aurangabad, and Delhi strains were resistant to DDT, yielding mortality rates of 90.9, 87.4, and 44.4% and 70.1, 3.0, and 6.0% for male and female adults, respectively. In contrast, adults of all three strains were found to be susceptible to malathion, deltamethrin, and permethrin, exhibiting mortalities between 98 and 100%. The magnitudes of susceptibility, based on the KDT50 values, were greater in the OX513A strain, as compared to wild-type strains of Ae. aegypti for all insecticides tested. The results confirm that, aside from historical resistance to DDT, OX513A has retained full sensitivity to these commonly used compounds and exhibits responses akin to those of susceptible Indian wild-type strains.

Optimization of a nonviral transfection system to evaluate Cox-2 controlled interleukin-4 expression for osteoarthritis gene therapy in vitro.

Gene therapy appears to have the potential for achieving a long-term remedy for osteoarthritis (OA). However, there is a risk of adverse reactions, especially when using cytomegalovirus-controlled expression. To provide a safe application, we focused on the expression of therapeutic cytokines [e.g. interleukin (IL)-4] in a disease-responsive manner by use of the previously cloned Cox-2 promoter as 'genetic switch'. In the present study, we report the functionality of a controlled gene therapeutic system in an equine osteoarthritic cell model. Different nonviral transfection reagents were tested for their efficiency on equine chondrocytes stimulated with equine IL-1β or lipopolysaccharide to create an inflammatory environment. To optimize the transfection, we successfully redesigned the vector by excluding the internal ribosomal entry site (IRES). The functionality of our Cox-2 promoter construct with respect to expressing IL-4 was proven at the mRNA and protein levels and the anti-inflammatory potential of IL-4 was confirmed by analyzing the expression of IL-1β, IL-6, IL-8, matrix metalloproteinase (MMP)-1, MMP-3 and tumor necrosis factor (TNF)-α using a quantitative polymerase chain reaction. Nonviral transfection reagents yielded transfection rates from 21% to 44% with control vectors with and without IRES, respectively. Stimulation of equine chondrocytes resulted in a 20-fold increase of mRNA expression of IL-1β. Such exogenous stimulation of chondrocytes transfected with pNCox2-IL4 led to an increase of IL-4 mRNA expression, whereas expression of inflammatory mediators decreased. The timely link between these events confirms the anti-inflammatory potential of synthesized IL-4. We consider that this approach has significant potential for translation into a useful anti-inflammation therapy. Molecular tools such as the described therapeutic plasmid pave the way for a local-controlled, self-limiting gene therapy.

A Killer–Rescue system for self-limiting gene drive of anti-pathogen constructs

A number of genetic mechanisms have been suggested for driving anti-pathogen genes into natural populations. Each of these mechanisms requires complex genetic engineering, and most are theoretically expected to permanently spread throughout the target species' geographical range. In the near term, risk issues and technical limits of molecular methods could delay the development and use of these mechanisms. We propose a gene-drive mechanism that can be self-limiting over time and space, and is simpler to build. This mechanism involves one gene that codes for toxicity (killer) and a second that confers immunity to the toxic effects (rescue). We use population-genetic models to explore cases with one or two independent insertions of the killer gene and one insertion of the rescue gene. We vary the dominance and penetrance of gene action, as well as the magnitude of fitness costs. Even with the fitness costs of 10 per cent for each gene, the proportion of mosquitoes expected to transmit the pathogen decreases below 5 per cent for over 40 generations after one 2 : 1 release (engineered : wild) or after four 1 : 2 releases. Both the killer and rescue genes will be lost from the population over time, if the rescue construct has any associated fitness cost. Molecular approaches for constructing strains are discussed.