EEB 180: Genetic Engineering in Developing Countries
GM mosquitoes for disease control
- Mosquito-borne diseases kill over 1,000,000 people every year.
- Among other control methods, there is an interest in engeineering mosquitoes with genes conferring refractoriness to the malaria parasite.
- Research is quickly progressing in the discovery of these genes.
- In addition, large-scale population replacement with refractory mosquitoes will require a gene drive system.
- Several drive systems are known that:
- show non-Mendelian patterns of inheritance; and
- are able to spread in the presence of a fitness cost.
- Research is also quickly progressing in the area of gene drive.
- In addition to these issues, ethical and ecological factors must also be considered.
- Several methods are available for generating refractoriness to malaria parasites within mosquitoes.
- Synthetic proteins have been engineered that interrupt the parasite's reproductive pathway in the mosquito.
- Antibodies have been discovered that kill parasites within the mosquito.
- Genes have been discovered that govern refractoriness in natural populations.
- Research is continuing in each of these directions.
Transgene confering refractoriness to rodent malaria:
- Engineered by the laboratory of Marcelo Jacobs-Lorena at Johns Hopkins University.
- Identified receptor sites for proteins that the parasite requires to pass through the gut after ingestion.
- Produced small proteins that saturate these receptor sites.
- This blocks amplification and transmission of the parasite in the mosquito.
- This research was conducted for P. berghei in An. stephensi.
- Future research should be conducted for P. falciparum in An. gambiae.
Figure 1: Mosquitoes become infected with the malaria parasite upon taking an infected human blood-meal. This produces an oocyst in the mosquito's gut wall (light red). When the oocyst ruptures, it releases sporozoites that pass through the gut (dark red) and into the hemocoel (white). The sporozoites are then amplified and migrate through the mosquito's body to the salivary glands, ready to infect a new human. Right: The laboratory of Marcelo Jacobs-Lorena at Johns Hopkins University has identified receptor sites for proteins that are necessary for the malaria parasite to pass through the gut wall after the oocyst ruptures. The same receptors are involved with the passage of sporozoites into the salivary glands. The laboratory has produced small proteins that preferentially occupy these sites (blue), blocking transmission of sporozoites through the gut wall and into the salivary glands. The appropriate gene constructs have been introduced into An. stephensi mosquitoes, thus rendering them refractory to P. berghei (a model system for human malaria).
Requirements of gene drive systems:
- Drive mechanism must be strong enough to spread anti-pathogen genes to fixation (or close to fixation) on a public health timescale.
- Tight linkage between the drive mechanism and anti-pathogen gene.
- Ability to spread large multi-gene constructs (it is more difficult for the pathogen to evolve resistance to multiple anti-pathogen genes).
- Ability to function in several vector species.
- Safe (doesn't cause undesirable effects in vector or non-target species).
- Ability to remove the anti-pathogen gene from the population in the event of unanticipated negative effects.
- One of the first gene drive systems to gain widespread attention.
- TEs replicate within a host genome and hence are inherited more frequently in the offspring’s genome.
- The increase in inheritance enables TEs to spread even in the presence of a fitness cost to the host.
- This has led to their widespread prevalence among many taxa.
- Various families of TEs represent 47% of the Aedes aegypti mosquito genome.
Figure 2: Two mechanisms by which Class II TEs can replicate. (A) In templated gap repair, excision and transposition leaves a gap that is sometimes sealed by copying information on the homologous chromosome. (B) In S-phase transposition, a replicated TE transposes to an unreplicated part of the genome and is replicated again.
Sources of encouragement:
- The P element spread through most of the global Drosophila melanogaster population within the span of a few decades following a natural acquisition from Drosophila willistoni.
- Hope that such an invasion could be repeated with an engineered TE in mosquito species.
- Failure to introduce a highly active TE into An. gambiae.
- Reduction of replication rate with increasing TE copy number (TEs do this to avoid corrupting the host genome).
- Accumulation of mutations within TEs leading to their inactivation (this is a form of reduction in replication and may make it hard to find a highly active TE).
- Decline in TE activity with increasing size.
- Vulnerability of TEs to losing internal sequences during replication.
- HEGs express an endonuclease that creates a double-stranded break at a highly specific site lacking the HEG.
- Homologous DNA repair then copies the HEG to the cut chromosome.
- This increases the representation of the HEG over subsequent generations.
Figure 3: A HEG is found between two specific sequenes of DNA. The HEG produces an endonuclease which cleaves a specific recognition sequence when it is not already filled by another HEG. This gap is then repaired using the homologous chromosome as a template, leading to another copy of the HEG.
Attractive features of HEGs:
- Capable of population invasion from a very low initial frequency.
- Restricted to one genomic location (therefore more stable than TEs).
- Very invasive following an accidental release.
- Possibility of losing inserted (anti-pathogen) DNA during gap repair.
- Natural HEGs have only been discovered in fungi, plants, bacteria and bacteriophages.
- Would need to be substantially modified before being used in insects.
- Engineer a HEG to target essential genes.
- When the HEG replicates, it produces a homozygous lethal mutation.
- This can be used to induce a population crash.
- However, there will be strong selection pressure for the evolution of suppressors.
- To investigate whether HEGs can operate in D. melanogaster and mosquitoes disease vectors.
- Attracted much attention following the observation that an engineered Medea element is able to rapidly spread through D. melanogaster populations in the laboratory.
- This synthetic element is based on a naturally-occurring selfish genetic element first discovered in the flour beetle Tribolium castaneum.
- Medea is able to rapidly spread through a population in the presence of a fitness cost by distorting the offspring ratio in its favor.
- It achieves this by causing the death of all offspring of heterozygous females that do not inherit the allele.
Synthetic Medea element:
- The synthetic Medea element works by the hypothesis that Medea encodes both a maternally-expressed toxin and a zygotically-expressed antidote.
- The toxin causes the death of all progeny lacking the Medea allele.
- The antidote rescues Medea-bearing progeny from an otherwise imminent death.
- In this way, the proportion of Medea-bearing individuals is increased with each generation.
Figure 4: A punnet square representing the reproductive advantage of the Medea allele. Offspring of females who do not inherit the Medea allele are killed by the maternal toxin because they are not able to express the zygotic antidote. This distorts the offspring ratio in favor of the Medea allele.
Attractive features of Medea:
- Medea spread is not retarded by the insertion of foreign DNA.
- A solution has been proposed to minimize the rate of dissociation of refractory genes.
- In the event that a refractory gene should be recalled following an environmental release; another strain of Medea can be introduced to replace the first.
- Medea is very likely to go extinct following a small accidental release but will spread quickly following an intentional release.
- To construct Medea systems for mosquitoes disease vectors.
The large number of species of malaria parasites and vectors makes malaria difficult to control genetically:
- Malaria is transmitted by any of five species of Plasmodium (P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi).
- These are transmitted to humans by ~50 species of mosquitoes (belonging to the genus Anopheles).
- The majority of deaths due to malaria in sub-Saharan Africa are due to P. falciparum transmitted by An. gambiae.
- But even if all An. gambiae mosquitoes are to be genetically modified, malaria will still remain prevalent because people will still receive several infective bites per year from other malaria vectors.
- If the strategy is to be successful, several species of mosquito vectors may need to be engineered.
Even within An. gambiae populations there are barriers to gene flow that must be considered:
- One of the major factors in the ecology of An. gambiae is the existence of at least five chromosomal forms that may be partially or totally reproductively isolated.
- These chromosomal forms (with the exception od the Forest form) are distinguished on the basis of six paracentric inversions on chromosome 2.
- Each of these chromosomal forms has its own distinct geographical distribution.
- In cases where these distributions overlap, gene flow between forms is limited.
- As an additional level of complication, there are also two molecular forms of An. gambiae whose distributions often coincide with those of the chromosomal forms.
- A significant understanding of the population structure of An. gambiae is essential to the success of a transgenic release.
Figure 5: Geographic population structure of An. gambiae throughout Africa showing the distribution of chromosomal inversions and molecular forms.
- It is unlikely that GM mosquitoes will provide an all-in-one solution.
- GM mosquitoes should be considered as a potential future ingredient of an integrated vector management strategy.
- This should include:
- Insecticide-treated bed-nets;
- Indoor residual spraying with insecticides; and
- Removal of mosquito breeding sites.
- Charlesworth, B., P. Sniegowski, and W. Stephan, 1994 The evolutionary dynamics of repetitive DNA in eukaryotes. Nature 371: 215-220.
- Chen, C. H., H. Huang, C. M. Ward, J. T. Su, L. V. Schaeffer et al., 2007 A sythetic maternal-effect selfish genetic element drives population replacement in Drosophila. Science 316: 597-600.
- Marshall, J. M., and C. E. Taylor, 2009 Transgenic mosquitoes for malaria control. PLoS Medicine 6: e1000020.
- Sinkins, S. P., and F. Gould, 2006 Gene drive systems for insect disease vectors. Nat. Rev. Genet. 7: 427-435.