The Genedrive

Presenter: David Youssef

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The Malaria Problem

• Progress against malaria had already stalled since 2016 as foreign donors drifted away. But 2020 is likely to be the first year in decades to see an increase in deaths, WHO warned in its 2020 World Malaria Report.1
• “It’s likely that excess malaria mortality is larger than direct covid-19 mortality,” said Pedro Alonso, director of WHO’s malaria programme. Malaria killed 409 000 people in 2019 and 411 000 in 2018, most of them babies and toddlers in sub-Saharan Africa.
• The total number of covid-19 deaths recorded so far in sub-Saharan Africa is just under 30 000, of which more than two thirds occurred in South Africa.

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Malaria Life Cycle

• The natural history of malaria involves cyclical infection of humans and female Anopheles mosquitoes.
• In humans, the parasites grow and multiply first in the liver cells and then in the red cells of the blood. In the blood, successive broods of parasites grow inside the red cells and destroy them, releasing daughter parasites (“merozoites”) that continue the cycle by invading other red cells.
• The blood stage parasites are those that cause the symptoms of malaria.
• When certain forms of blood stage parasites (gametocytes, which occur in male and female forms) are ingested during blood feeding by a female Anopheles mosquito, they mate in the gut of the mosquito and begin a cycle of growth and multiplication in the mosquito.
• After 10-18 days, a form of the parasite called a sporozoite migrates to the mosquito’s salivary glands.
• When the Anopheles mosquito takes a blood meal on another human, anticoagulant saliva is injected together with the sporozoites, which migrate to the liver, thereby beginning a new cycle.
• Thus the infected mosquito carries the disease from one human to another (acting as a “vector”), while infected humans transmit the parasite to the mosquito,
• In contrast to the human host, the mosquito vector does not suffer from the presence of the parasites.

https://www.cdc.gov/malaria/about/biology/index.html

What is a Genedrive?

• In nature, certain genes ‘drive’ themselves through populations by increasing the odds that they will be inherited.
• Examples include:
• Endonuclease genes that copy themselves into chromosomes lacking them,
• Segregation distorters that destroy competing chromosomes during meiosis,
• Transposons that insert copies of themselves elsewhere in the genome,
• Medea elements that eliminate competing siblings who do not inherit them,
• Heritable microbes such as Wolbachia .
• Natural homing endonuclease genes exhibit drive by cutting the corresponding locus of chromosomes lacking them.
• This induces the cell to repair the break by copying the nuclease gene onto the damaged chromosome via homologous recombination
• The copying process is termed ‘homing’, while the endonuclease-containing cassette that is copied is referred to as a ‘gene drive’ or simply a ‘drive’.
• Because copying causes the fraction of offspring that inherit the cassette to be greater than 1/2 these genes can drive through a population even if they reduce the reproductive fitness of the individual organisms that carry them.
• Over many generations, this self-sustaining process can theoretically allow a gene drive to spread from a small number of individuals until it is present in all members of a population.

What is a Genedrive? (cont.)

What is the biochemically happening?

a | An X-chromosome shredder (X-shredder) system works by expressing an endonuclease from the Y chromosome, in an X-Y heterogametic species, that cleaves the X chromosome at many locations. This destroys the X chromosome, so all viable sperm have only Y chromosomes, leading to all male offspring and, eventually, population suppression. A red background denotes lethality.

b | An RNA-guided CRISPR (clustered regularly interspaced short palindromic repeats) endonuclease with one or more small guide RNAs (gRNAs) may be used as the X-shredder.

c | An RNA-guided endonuclease X-shredder can be reversed using an X chromosome containing multiple gRNAs targeting the gRNAs of the original X-shredder. These X-chromosome-localized gRNAs would be activated before the gRNAs on the Y chromosome, resulting in removal of the gRNAs on the Y chromosome before the X chromosome is shredded. This permanently inactivates the X-shredder, resulting in increased production of female offspring, which have a major fitness advantage compared to male offspring when X-shredder alleles remain in the population.

https://www.nature.com/articles/nrg.2015.34/figures/3

What is the Genetically happening?

a | A homing endonuclease gene (HEG) works by encoding an endonuclease, which cleaves at a target site on the homologous chromosome opposite the HEG. Homology-directed repair (HDR) results in the HEG being copied to the homologous chromosome.

b | A homing element may be generated using an RNA-guided CRISPR (clustered regularly interspaced short palindromic repeats) endonuclease together with one or more small guide RNAs (gRNAs). Resistance alleles can be minimized by targeting the homing-based RNA-guided drive to a conserved critical gene at multiple locations using several gRNAs. The gene would only be reformed to functionality if HDR takes place, precluding successful repair and induction of resistance alleles by non-homologous end joining (NHEJ).

c | A homing-based RNA-guided drive may be removed from a population by designing a reversal drive encoding a gRNA that targets the previous generation drive. d | A homing drive may be utilized to suppress a population by homing into a critical gene, the disruption of which induces recessive sterility (in this example, female infertility) or lethality.

https://www.nature.com/articles/nrg.2015.34/figures/1

How does this play out in a population?

a | A homing drive results in most or all progeny of heterozygotes receiving the homing element, which allows the drive to spread rapidly throughout the population.

b | A second-generation reversal drive can overwrite a first-generation homing drive, replacing its payload gene. Progeny of heterozygotes with this drive will all inherit the second-generation drive. This homing drive may be configured to home into wild-type alleles as well, immunizing the population against the first-generation homing drive.

c | A suppression drive targeting a recessive gene required for viability or fertility will spread rapidly from heterozygotes with the drive, but would create an increasing number of sterile or unviable homozygotes, eventually resulting in a population crash.

https://www.nature.com/articles/nrg.2015.34/figures/2

Value Proposition

• A team of researchers writing in the journal Nature Communications has shown that a gene drive can be used to suppress infection with cytomegalovirus, a type of herpesvirus.
• The underlying molecular mechanism of the gene drive is similar to others before it: A self-propagating chunk of DNA inserts itself into a gene that is important to the virus.
• In this case, the gene is UL23, which is needed for cytomegalovirus to avoid the human immune response.
• The researchers showed that when a cell is infected by both the normal virus (called "wildtype" or "WT") and the modified virus carrying a gene drive ("GD"), the gene drive was able to quickly and efficiently spread through the entire population, representing up to 95% of the final proportion of viruses.
• The end result is the suppression of viral infection (in cell culture, not in an animal model) because the gene drive virus lacks the important UL23 gene, which is needed for the virus to avoid a potent immune molecule known as interferon gamma (IFN-γ), which the authors added to the cell culture.

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https://www.acsh.org/news/2020/09/30/gene-drives-could-kill-mosquitoes-and-suppress-herpesvirus-infections-15060

Security Risks

• The JASONs, a group of elite scientists that advises the US government on national security, in June, the secretive group took stock of a new threat: gene drives, a genetic-engineering technology that can swiftly spread modifications through entire populations and could help vanquish malaria-spreading mosquitoes.
• That meeting forms part of a broader US national security effort this year to grapple with the possible risks and benefits of a technology that could drive species extinct and alter whole ecosystems. On 19 July, the US Defense Advanced Research Projects Agency (DARPA) announced US$65 million in funding to scientists studying gene-editing technologies; most of the money will be for work on gene drives.
• A US intelligence counterpart to DARPA is planning to fund research into detecting organisms containing gene drives and other modifications.
• Teams receiving military funding also plan to develop tools to counter rogue gene drives that spread out of control. Such methods include chemicals that block gene-editing or ‘anti-gene drives’ that can reverse a genetic modification or immunize unaltered wild organisms so they are resistant to a gene drive.
• These tools could combat a gene drive deployed to do harm, such as those that engineer insects to transmit diseases more effectively or deliver toxins. But such countermeasures are far more likely to be deployed against accidental gene-drive releases from research labs.
• Lax or non-existent biosafety guidelines for working on gene-drive organisms increase the odds of a release. Other efforts are afoot to fund work studying the national security implications of gene drives.

Toward Risk Management

(i) Before any primary drive is released in the field, the efficacy of specific reversal drives should be evaluated. Research should assess the extent to which the residual presence of guide RNAs and/or Cas9 after reversal might affect the phenotype or fitness of a population and the feasibility of reaching individual organisms altered by an initial drive.

(ii) Long-term studies should evaluate the effects of gene drive use on genetic diversity in target populations. Even if genome-level changes can be reversed, any population reduced in numbers will have reduced genetic diversity and could be more vulnerable to natural or anthropogenic pressures. Genome-editing applications may similarly have lasting effects on populations owing to compensatory adaptations or other changes.

(iii) Investigations of drive function and safety should use multiple levels of molecular containment to reduce the risk that drives will spread through wild populations during testing. For example, drives should be designed to cut sequences absent from wild populations, and drive components should be separated.

(iv) Initial tests of drives capable of spreading through wild populations should not be conducted in geographic areas that harbor native populations of target species.

(v) All drives that might spread through wild populations should be constructed and tested in tandem with corresponding immunization and reversal drives. These precautions would allow accidental releases to be partially counteracted.

Toward Risk Management (cont.)

(vi) A network of multipurpose mesocosms and microcosms should be developed for testing gene drives and other advanced biotechnologies in contained settings.

(vii) The presence and prevalence of drives should be monitored by targeted amplification or metagenomic sequencing of environmental samples.

(viii) Because effects will mainly depend on the species and genomic change rather than the drive mechanism, candidate gene drives should be evaluated on a case-by-case basis.

(ix) To assess potentially harmful uses of drives, multidisciplinary teams of experts should be challenged to develop scenarios on deliberate misuse.

(x) Integrated benefit-risk assessments informed by the actions recommended above should be conducted to determine whether and how to proceed with proposed gene drive applications. Such assessments should be conducted with sensitivity to variations in uncertainty across cases and to reductions in uncertainty over time.

Containment Strategies

• Ecological containment involves building and testing gene drives in geographic areas that do not harbor native populations of the target species.
• For example, most gene drive studies involving tropical malarial mosquitoes have been conducted in temperate regions in which the mosquitoes cannot survive or find mates.
• Molecular containment ensures that the basic requirements for drive are not met when mated with wild-type organisms.
• True drives must cut the homologous wild-type sequence and copy both the gene encoding Cas9 and the guide RNAs.
• Experiments that cut transgenic sequences absent from wild populations and copy either the gene encoding Cas9 or the guide RNAs - but not both - should be quite safe. Ecological or molecular containment should allow basic research into gene drive effectiveness and optimization to be pursued with negligible risk

References

1. Champer, J., Buchman, A. & Akbari, O. Cheating evolution: engineering gene drives to manipulate the fate of wild populations. Nat Rev Genet 17, 146–159 (2016). https://doi.org/10.1038/nrg.2015.34
2. Callaway, E. US defence agencies grapple with gene drives. Nature 547, 388–389 (2017). https://doi.org/10.1038/nature.2017.22345
3. Oye, K. A., Esvelt, K., Appleton, E., Catteruccia, F., Church, G., Kuiken, T., Lightfoot, S. B.-Y., McNamara, J., Smidler, A., & Collins, J. P. (2014). Regulating gene drives. Science, 345(6197), 626–628. https://doi.org/10.1126/science.1254287
4. Esvelt KM, Smidler AL, Catteruccia F, Church GM. Concerning RNA-guided gene drives for the alteration of wild populations. Elife. 2014;3:e03401. Published 2014 Jul 17. doi:10.7554/eLife.03401

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