Self-sustaining

Our long-term goal is to produce modified mosquitoes that are fertile and that can pass on to their progeny a change in their genes that is self-sustaining. We are currently investigating two options: 1) fertile males carrying a gene that will spread through the mosquito population and as it does so causes a male bias sex ratio; 2) fertile males carrying a gene that will spread through the mosquito population and as it does so causes females that inherit the gene from both parents to be sterile. Both approaches would lead to a reduction in the malaria mosquito population. While these approaches are different, they may not be mutually exclusive.

Timeline

The initial steps of our development pathway are the same as for the sterile males and the self-limiting male bias. But if our tests and evaluations advance sufficiently, and we feel confident that we have a viable technology that could be used to fight malaria, then our development pathway will be different from the previous phases.

Because we need to assume that the genetic modification could persist and spread in the environment in Africa, we need to think differently about the stages of research that takes place in the region where these mosquitoes are native.

As we are pioneering the developmental pathway for self-sustaining modified mosquitoes there are many consultation and evaluation steps that we need to take and these may also change over time. These currently will include evaluation by the World Health Organisation (WHO) Vector Control Advisory Group and other groups at the WHO (e.g the vector control pre-qualification team) before and after release, as well as comprehensive independent ecological and socio-economic risk assessments, community and stakeholder support.

What Are Our Approaches

Biasing the Sex Ratio

This strategy relies on altering the sex ratio in malaria mosquito populations to decrease the number of female malaria mosquitoes relative to males. Only female Anopheles gambiae transmit the disease, and a reduction in the number of females limits reproduction and the future population size, therefore reducing the number of vectors for malaria

The approach is based around the sex-determining chromosomes (XY for males, and XX for females) and relies on the fact that female offspring require two functional X chromosomes– one from each parent – in order for female offspring to be produced.

Target Malaria researchers have used nuclease enzymes (image 1) to identify and cut through several key sites on the X chromosome (image 2) in the sperm of male Anopheles gambiae which leads to a fragmentation of this chromosome (image 3). When these males reproduce, they can still pass on a functional Y chromosome to their offspring, but they cannot pass on a functional X chromosome due to its fragmentation (image 4). This results in a bias toward male (XY) offspring.

Work published by our team in June 2014 has shown that we can successfully distort the sex ratio of a laboratory population, as over 95% of the offspring produced by modified Anopheles gambiae were male, with only 5% being female. By comparison, under normal circumstances, researchers would expect a 50:50 split between male and females, meaning that our modification reduces the number of females produced by 10-fold.

Mathematical models indicate that the approach could be highly effective in reducing mosquito numbers. Because the modification is intended to be carried on the Y chromosome, it would also be self-sustaining. The male offspring of modified males would remain fertile and every single one of them would have the sex-distorting nuclease gene on their Y chromosome, allowing them to continue the trend of producing mostly male offspring.

Biasing the Sex Ratio

  • To help reduce the population of malaria mosquitoes, Target Malaria researchers are looking at several strategies. One is focused on biasing the sex ratio. Target Malaria researchers have identified several key sites on the X chromosome that are unique and not present in the Y chromosome.
  • A nuclease gene that produces a nuclease enzyme is placed on the Y chromosome. It recognises the key sites on the X chromosome, cutting through them and leaving the chromosome fragmented. The Y chromosome remains intact.
  • Individuals that inherit an X chromosome from their father are female. Individuals that inherit a Y chromosome from their father are male. Because the males now have fragmented X chromosomes, they can only produce male offsprings. This is called sex biasing.
  • Target Malaria scientists modify male mosquitoes to carry the nuclease gene and then release them into the wild population.
  • During reproduction with a wild female, the modified males can only pass on a functional Y chromosome. This will result in up to 100% male offspring. The male offsprings carry the genetic modification and will pass it on through future matings.
  • Over time, as more modified males mate with wild females, there will be more male mosquitoes in the population and fewer females, leading to a significant reduction in the overall size of the population and the number of mosquitoes able to transmit malaria. Studies have shown that sex-biasing can result in a population in which only 5% of all mosquitoes are females.

Focusing on Mosquito Female Fertility

This strategy focuses on using nucleases to knock out genes that are key to fertility in female Anopheles gambiae mosquitoes. The approach could significantly reduce the prevalence of malaria because the number and productivity of females in a population determines future population size, and female Anopheles gambiae are highly effective vectors for the disease.

In order to knock out female fertility genes, the nucleases are designed to identify the specified genes (image 1) and cut through them (image 2). When this stretch of DNA is repaired, the nuclease gene is copied and inserted into the cut site (image 3), interrupting the original gene and preventing it from working properly (image 4).

A female that has one copy of this fertility gene disrupted will be able to reproduce normally, but when both copies within her chromosomes are disrupted, the female cannot produce viable offspring.

We have designed these nucleases so that they are only active in the cells of the mosquito that make the sperm and the eggs. Due to the preferential copying mechanism of these nuclease genes in the sperm and eggs, an individual initially containing only one copy of the gene will transmit it to many more offspring than normal.

As fertility genes are fully disrupted in females that inherit two copies of the nuclease gene, this should lead to an overall reduction in the population.

Reducing Female Fertility

  • Target Malaria scientists introduce a nuclease that disrupts a gene essential for female fertility, causing females to be sterile when both copies of the gene are disrupted. The modified male and female each carry one copy of a chromosome with the nuclease gene and are fertile. The gene must be present on both copies of a female’s chromosomes in order to cause infertility.
  • During the production of eggs and sperm, the gene produces an enzyme that cuts the unmodified chromosome and "homes" into the female fertility gene
  • As the second chromosome repairs itself, it copies the genetic material from the modified chromosome. In the cells that make the eggs and the sperm, both chromosomes now carry the nuclease gene. With both copies of the chromosome carrying the gene, all sperm and eggs will receive a copy of the nuclease and the modification has a 100% chance of being passed to offspring.
  • Male mosquitoes that have been successfully modified in the lab are released into wild populations.
  • When they mate with wild females, they pass on the nuclease gene, resulting in 100% of offspring carrying the nuclease of the gene that causes infertility in females.
  • The offspring of the modified males and wild females each carry one copy of the gene, and will replicate the nuclease homing process during the production of their own eggs and sperm.
  • Over time, the gene will pass to enough members of the mosquito population that males and females who each carry a copy of the gene will mate. When two modified mosquitoes mate, their offspring will receive two copies of the modified chromosome,resulting in infertility in the females.
  • Males who inherit two copies of the gene can still reproduce and pass the nuclease to future generations. As the population of female mosquitoes becomes increasingly infertile, the population of malaria mosquitoes should be reduced.