Question 2: What types of engineered gene drives that the additional voluntary guidance materials should focus on?

Argentina doesn´t agree in create additional voluntary guidance materials for LMO containing gene drives.

--- Posted on behalf of Dr. Luciana Pimenta Ambrozevicius, Brazil ---

Dear Participants,

For the low-threshold gene-drive systems the two primary strategies are referred to as population suppression, that are the genetic equivalent of insecticides, and the population modification or replacement, that leaves the insect in place but blocks disease transmission. 

The guidance material could possibly focus on the different mechanisms of suppression drives that have the greatest impact on disease transmission on a continental scale. For example, the suppression of Aedes aegypti would greatly reduce the transmission of all arboviruses vectored by this species, (dengue fever, yellow fever, chikungunya and Zika).

Are there other engineered gene drive systems that could also be considered?

Luciana

The positive and negative impacts of engineered gene drives on food chains, within populations, species and other communities, ecosystems, ecological niches, Mother Nature and environmental are not well understood in IPLCs. The science and technology, innovations related LMO, containing engineered gene drive and their different types are being difficult to follow and access information. The genetic expressions and its functions in biological systems of species while use of the engineered gene drives and its future impacts are unknown.

A clear information systems, inclusive, participatory and full and effective participation mechanism of IPLCs, regulatory and moratorium, with customary systems and rights of IPLCs in the voluntary guidance materials.

Kamal Kumar Rai
IPLCs

Hello everybody, my name is Christoph Then and I am a member of ENSSER (The European Network of Scientists for Social and Environmental Responsibility) and representing Testbiotech (http://www.testbiotch.org) in this discussion. Testbiotech compiled recent information in a backgrounder for this forum https://www.testbiotech.org/node/3037. In our backgrounder we suggest to widen the perspective to integrate not only engineered gene drives, but all LMOs with self-propagating artificial genetic elements (SPAGE) that are intended to actively spread technically inserted genetic elements within domesticated or non-domesticated populations.

The types of LMOs containing engineered gene drives that could be considered can include those aimed at controlling disease vectors of public health importance such as mosquitoes (e.g. Malaria); LMOs containing engineered gene drives that may be used to eradicate invasive species, e.g. Mus musculus (the house mouse which is a serious problem in Marion Island), water hyacinth; as well as LMOs containing engineered gene drives that could be used to control agriculture pests of economic importance, e.g. Spodoptera frugiperda (Fall armyworm).

Many different types of gene drive systems have been discussed as suitable for use in mosquitoes and other species. Lists and brief descriptions of the approaches which have garnered the greatest interest to date can be found in the studies below.

• Gene drives to fight malaria: current state and future directions (Hammond & Galizi, 2017) Available at https://www.tandfonline.com/doi/full/10.1080/20477724.2018.1438880; 
• Adequacy and sufficiency evaluation of existing EFSA guidelines for the molecular characterisation, environmental risk assessment and post‐market environmental monitoring of genetically modified insects containing engineered gene drives (EFSA, 2020) Available at https://www.efsa.europa.eu/en/efsajournal/pub/6297;
• Standardizing the definition of gene drive (Alphey et al., 2020) Available at https://www.pnas.org/doi/10.1073/pnas.2020417117;
• Combating mosquito-borne diseases using genetic control technologies (Wang et al, 2021) Available at https://www.nature.com/articles/s41467-021-24654-z;
• A male-biased sex-distorter gene drive for the human malaria vector Anopheles gambiae (Simoni et al., 2020) Available at https://www.nature.com/articles/s41587-020-0508-1;
• Efficient population modification gene-drive rescue system in the malaria mosquito Anopheles stephensi (Adolfi et al., 2020) Available at https://www.nature.com/articles/s41467-020-19426-0;
• Gene drive designs for efficient and localisable population suppression using Ylinked editors (Geti et al. 2022) Available at https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010550;
• Driving down malaria transmission with engineered gene drives (Garrood et al. 2022) Available at https://www.frontiersin.org/articles/10.3389/fgene.2022.891218/full;
• Double drives and private alleles for localised population genetic control. (Willis K. & Burt A., 2021) Available at https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1009333;
• Next-generation gene drive for population modification of the malaria vector mosquito, Anopheles gambiae (Carballar-Lejarazu R. et al., 2020) Available at https://www.pnas.org/doi/full/10.1073/pnas.2010214117;
• Gene drives gaining speed (Bier, 2022) Available at https://www.nature.com/articles/s41576-021-00386-0;
• Closing the gap to effective gene drive in Aedes aegypti by exploiting germline regulatory elements (Anderson et al. 2023) Available at https://www.nature.com/articles/s41467-023-36029-7;
• The Challenges in Developing Efficient and Robust Synthetic Homing Endonuclease Gene Drives (Verkuijl et al. 2022) Available at https://www.frontiersin.org/articles/10.3389/fbioe.2022.856981/full#h6

Sincerely

Dear colleagues,
My name is Werner Schenkel, I am working in risk assessment and risk management for the Germany competent authority and follow the discussions on gene drive organisms and Cartagena Protocol activities since about 2015.
Taking into account general considerations of LMO containing gene drives, as requested in Decision CP 10/10, needs to include considering all types of engineered gene drives that may have the potential to cause adverse effects on biodiversity.
Although any risk assessment must be performed on a case by case basis, a classification of gene drives within guidance materials will help to identify challenges in risk assessment that may be specific to all or only for some gene drives. Moreover, it will help to identify challenges in the risk assessment of LMO containing engineered gene drives that may be shared with other LMO or even other human interventions (e.g. introduction of alien taxa s. #11559).
Reasonable classifications of types of gene drives have been presented in several publications. A sound description and explanation is given in the WHO guidance framework for testing genetically modified mosquitoes (https://apps.who.int/iris/bitstream/handle/10665/341370/9789240025233-eng.pdf?sequence=1&isAllowed=y). Here gene drives are classified according to:
• strategy (populations suppression vs. population modification (replacement))
• temporal characteristics (self-limiting vs. self-sustaining)
• spatial characteristics (localizing vs. non localising).
In other publications (EFSA GMO Panel: https://www.efsa.europa.eu/en/efsajournal/pub/6297, Devos et al. 2020: https://doi.org/10.1080/07388551.2021.1933891) localizing or non-localising is replaced by high- vs. low-threshold. The later may give a closer connection to the technical implementation of the desired localisation as well as a relation to other risk related considerations.
Besides these “phenotypic” classifications, gene drives can be grouped according to the molecular mechanisms to achieve the drive (e.g. homing vs. non-homing based). As the mechanism is generally correlated to the “phenotypic” outcome these aspects are often used to subdivide above mentioned classes. It may still be reasonable to consider in guidance materials the different types of molecular mechanism independent of the desired outcome. It should be avoided to extrapolate properties of one type, e.g. homing based drives, to all types of gene drives.
Covering these different types of gene drives in additional voluntary guidance materials will allow for a more specific implementation of a case by case risk assessment.

Thanks very much to the moderator and contributors for this very interesting discussion.

As already shared on this thread by previous contributors, there are several ways to categorise GDOs that I do not need to repeat (e.g. self-sustaining versus self-limiting and so on).

However, an important concern regarding these categorisations is that they rely on intended outcomes, yet unintended outcomes can result in a blurring or even reversing of categories when unintended technical outcomes and/or wider real world release settings are taken into consideration. This has important implications for any reliance on these definitions, particularly for safety purposes.
1. Self-limiting drives have been suggested to potentially spread indefinitely under the right ecological conditions https://doi.org/10.1534/g3.120.401484
2. Homing drives have recently been discovered to be inadvertently functioning as meiotic drives due to unintended molecular effects that are leading to chromosomal loss https://doi.org/10.1101/2020.12.15.421271 https://doi.org/10.1038/s41467-021-21771-7
3. Modification drives have been shown to unintentionally result in population suppression https://doi.org/10.1371/journal.pgen.1008440
4. Split-drives have been shown to act as ‘shadow drives’ whereby they behave more like self-sustaining than self-limiting drives. Split drives are designed to have genetic components of a gene drive system separated, or ‘split’ across different chromosomes to limit inheritance of all components through subsequent populations. However, ‘shadow’ drives can be generated if mosquitoes, despite not inheriting the Cas9 gene, can still inherit the Cas9 protein via maternal deposition of the enzyme from the mother to the fertilized egg. Shadow drives have been documented in various studies:
https://doi.org/10.1111/mec.15788 https://doi.org/10.1534/g3.119.400985
https://doi.org/10.1038/s41467-021-21771-7 https://doi.org/10.1038/s41467-019-09694-w


The above data show a current lack of complete understanding of how gene drive technologies are working at a fundamental level, suggesting a high level of uncertainty regarding the basic science of gene drive techniques, and ecological dynamics could further blur these definitions.

Second, from the current evidence it would appear highly premature to rely on differential categories of gene drives that may limit risk assessment guidance to intended product design, without taking into account the potential for all unintended effects.

Thanks very much
Eva

Dear colleagues
Risk assessment should be conducted on a case-by-case basis, depending on the living modified organism concerned, its intended use, and the likely potential receiving environment.

The following trends/ applications for gene drives are at various stages of development (from laboratory experiments to environmental release) and can be considered for inclusion in the guidance:

- Controlling infectious diseases for human
i. Using genes drives to reduce the population of vectors and/or reservoirs or make them resistant to diseases. (E.g. Control malaria by suppressing Anopheles gambiae mosquitoes (vector) population using gene drive to cause sterility in females1 or by modifying the sex-determining gene, making the male gene dominant over the female one2, and tackling Lyme disease by making white-footed mice (reservoir) immune to Borrelia bacteria.)
ii. targeting infectious species without sexual reproduction to suppress viral infection3 or inactivate antibiotic resistance in bacteria4.

- Controlling invasive species & pests
i. Eradicate invasive vertebrates, like mice, rats, and rabbits, from islands by causing death or sterility in homozygous females or changing females into sterile males5.
ii. Spread a genetic alteration that re-sensitizes pests to toxins or makes them sensitive to otherwise innocuous compounds. E.g., reversing insecticide resistance in fruit flies6 or rendering weed susceptible to herbicides7.

- Adapting gene drives to protect endangered species or make crops resistant to disease.

1. Hammond A, Galizi R, Kyrou K, et al. A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nat Biotechnol. 2016;34(1):78-83.
2. Kyrou K, Hammond AM, Galizi R, et al. A CRISPR–Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes. Nat Biotechnol. 2018;36(11):1062-1066.
3. Walter M, Verdin E. Viral gene drive in herpesviruses. Nature Communications. 2020;11(1):4884.
4. Valderrama JA, Kulkarni SS, Nizet V, Bier E. A bacterial gene-drive system efficiently edits and inactivates a high copy number antibiotic resistance locus. Nature Communications. 2019;10(1):5726.
5. Prowse TAA, Cassey P, Ross JV, Pfitzner C, Wittmann TA, Thomas P. Dodging silver bullets: good CRISPR gene-drive design is critical for eradicating exotic vertebrates. Proc Biol Sci. 2017;284(1860).
6. Kaduskar B, Kushwah RBS, Auradkar A, et al. Reversing insecticide resistance with allelic-drive in Drosophila melanogaster. Nature Communications. 2022;13(1):291.
7. Neve P. Gene drive systems: do they have a place in agricultural weed management? Pest Manag Sci. 2018;74(12):2671-2679.

O.A.El-kawy

Hi, My name is Kemal Melih Taskin. I am a plant scientist working on plant development and biosynthetic pathways at Canakkale 18 Mart University, Turkiye. I am also a member of the scientific committee on Genetically Modified Organisms risk assessments.
For Gene Drive Technologies we require assessments that are flexible for the scientists to improve the technology and guidance for changes in public acceptance. In Turkiye, however, we have not prepared any guidelines for Gene drive technology risk assessments. I would be very grateful if anyone suggests how to start. Best wishes

I would also like to express appreciation to our moderator and again thank colleagues for their stimulating posts.

Our colleague Dr Agapito-Tenfen's comments in answer to Question 4 included a point that I believe should also appear in this thread, if it hasn't, and one which I would like to emphatically support. She said [#11615] "many other systems and organisms have been described as potential applications of gene drives to which such guidance might not fully apply." Guidance should be relevant to mosquitos and plants, of course, but also to gene drives that do not rely on the same relevant biology as plants and animals.

Gene drives have somehow become reformulated in recent years to be only those that manifest through sexual reproduction. Sexual reproduction through meiosis is but an example of a mechanism by which drives can spread and distort expected ratios. Since the 1950s, gene drives dependent on meiosis have been called “meiotic drives” because they use sexual reproduction to spread. This was discussed in the seminal 1957 review by the two great geneticists, Larry Sandler and Ed Novitski, who coined the term. Meiotic drives are a special case of the more generic gene drives. Their words:

“Instances are known both from studies of natural populations and laboratory experiments in which heterozygotes produce two kinds of gametes, not with the customary equality, but with unequal frequencies. Such meiotic behavior will profoundly affect gene frequencies in populations, and is referred to as meiotic drive.” L. Sandler and E. Novitski, "Meiotic Drive as an Evolutionary Force," The American Naturalist 91, no. 857 (Mar. - Apr., 1957): 105-110.

The use of gene drives in asexually reproducing organisms (or potentially in organisms that may switch between sexual and asexual, such as fungi and plants) is also discussed in the current biotechnology literature, such as in de Lorenzo, V. Microbial Biotechnology (2017)10(5), 995– 998. “The issue involves not only genetic assembly of all biological activities necessary to manifest the desired activity, but also its propagation through the environmental microbiome through either horizontal gene transfer or some type of prokaryotic gene drive, e.g. with engineered phages.”

This 2019 paper in Nature Communications says it plainly: “Here, we develop an analogous CRISPR-based gene-drive system for the bacterium Escherichia coli…This “proactive” genetic system (Pro-AG) functionally inactivates an antibiotic resistance marker on a high copy number plasmid…can likewise effectively edit large plasmids or single-copy genomic targets or introduce functional genes, foreshadowing potential applications to biotechnology or biomedicine.” https://doi.org/10.1038/s41467-019-13649-6 The paper is discussed in a 2022 review published in Nature Reviews Genetics https://doi.org/10.1038/s41576-021-00386-0 which says: “This study generalizes the concept of gene drive to bacteria, where it is applied to efficiently reduce the frequency of antibiotic resistance [sic].” That view is repeated in other prominent articles, such as https://doi.org/10.1002/bies.202100279.

Earlier this year a gene driven system based on bacterial conjugation was published in Applied and Environmental Microbiology https://doi.org/10.1128/aem.00053-23. “To resolve this problem, we envision a gene-driven system conferring biased inheritance to enable the delivery of a working plasmid without selection”.

Targets of gene drives may also include maternally inherited organelles or cytoplasmic symbionts that do not have a meiotic cycle even though the organelle is transmitted through differentiated gametes.

Best wishes to all
Jack

Dear colleagues,

The voluntary guidance materials should be useful and applicable for the case-by-case risk assessment of a variety of gene drive applications. Although a specific focus of the voluntary guidance materials should be on gene drive mosquitos, other applications under development or foreseen for the future should not be neglected, e.g. mammals like mice or rats, or other insect species.

In addition (and as also mentioned by others), the various types of gene drives should be considered (e.g. modification drive as well as suppression drives, local as well as global applications, threshold-dependent systems as well as low or no threshold gene drive) and different types of application (e.g. for vector control or applications in nature conservation).

Best regards,
Anita

Thank you moderator,

I am Josphat Muchiri, Deputy Director, Technical Services and BCH Focal Point, National Biosafety Authority, Kenya.

Additional guidance documents on gene drives should only be developed only in very unique circumstances where a general guidance document has limitations based on the ecology of the specific gene drive. For now, its recommended we test the test the guidance document being developed for mosquitoes, others mayl be considered in the future if they demonstrate unique characteristics, and based on the lessons learnt on mosquitoes. Its not feasible to develop a voluntary guidance any time a new gene drive is being developed.

The focus of the voluntary guidance material was decided to be quite specific. Nonetheless it should be kept in mind though, that for all case-by-case risk assessments general considerations specific for gene drives are important. When drafting the guidance enough thought should be given to those considerations, this is especially important since the development of LMOs containing engineered gene drives is a fast moving field with new technical concepts still emerging.

Dears,

We would like to share some additional thoughts to the very interesting points in this part of the forum.

For additional voluntary guidance materials to be proportionate, useful and practical, they may need to be tailored to the most likely cases gene drive modified organisms (GDMOs) moving to practical applications for “market” environmental release, requiring prioritisation of their scope and regular updates as priority applications change. This implies following a case-specific, stepwise and iterative approach, instead of trying to capture all possible hypothetical GDMO cases in “one go”. There is currently no direct experience conducting risk assessment for environmental releases of GDMOs. Therefore, the development process of additional voluntary guidance materials should involve a range of expert input including relevant stakeholders, be iterative, and build on a review of actual case studies by risk assessment experts. Once in place, regular review must be continued to ensure overall utility of additional voluntary guidance materials and their applicability, and to assess where any refinements are necessary. This may help to ensure that additional voluntary guidance materials are realistic and proportionate, and remain consistent with the weight of scientific evidence and familiarity gained with environmental releases of GDMOs and genetically modified organisms that do not contain an engineered gene drive.

As suggested by Brinda Dass in [#11582] and Werner Schenkel in [#11580], it would be helpful that additional voluntary guidance materials identify the broad types/categories of engineered gene drives currently explored in insects they will be applicable to. As mentioned by Brinda Dass in [#11582] and Werner Schenkel in [#11580], useful engineered gene drive classifications in insects have been reported in the following publications.

-2021 – Guidance Framework for testing of genetically modified mosquitoes – WHO – https://apps.who.int/iris/rest/bitstreams/1347934/retrieve
-2021 – Facilitating the conversation: Gene drive classification – Health Security – https://doi.org/10.1089/hs.2021.0109
-2021 – Potential use of gene drive modified insects against disease vectors, agricultural pests and invasive species poses new challenges for risk assessment – Critical Reviews in Biotechnology – http://dx.doi.org/10.1080/07388551.2021.1933891
-2021 – Gene Drive-Modified Organisms: Developing Practical Risk Assessment Guidance – Trends in Biotechnology – http://dx.doi.org/10.1016/j.tibtech.2020.11.015
-2021 – Engineering the composition and fate of wild populations with gene drive – Annual Review of Entomology – https://doi.org/10.1146/annurev-ento-020117-043154
-2020 – Adequacy and sufficiency evaluation of existing EFSA guidelines for the molecular characterisation, environmental risk assessment and post-market environmental monitoring of genetically modified insects containing engineered gene drives – EFSA Journal – http://dx.doi.org/10.2903/j.efsa.2020.6297
-2020 – Progress towards engineering gene drives for population control – Journal of Experimental Biology – https://doi.org/10.1242/jeb.208181

We hope the information shared will be helpful.

Wishing you all the best,

Yann Devos (Senior scientific officer in the Chief Scientist Office of the European Food Safety Authority (EFSA)) and Alexandre Huchelmann (Policy officer in the Biotechnology Unit of the Directorate-General for Health and Food Safety (DG SANTE) of the European Commission)

Dear participants

I´m Carolina Villafañe from The Ministry of Environment of Colombia, and I am in charge of Biosafety issues and, The Cartagena Protocol.

To determine what types of engineered gene drives the additional voluntary guidance materials should focus on, I recommend do not only focus on the gene drive design or mechanism, because one type of gene drive design or mechanism could be "relatively safe" or, "relatively risky", depending on, the host organism, the recipient organism, the objective of the LMO use, the receptor environment, etc. for these reasons the case by case and step by step principles continuing to be useful for the risk assessment.

According to the above, the types of engineered gene drives the additional voluntary guidance materials should focus on, should be the "gene drive-organism systems" identified with the methodology approximation I proposed in the intervention #11631.

I mean, focusing on the type of gene drive (technology by itself), would not be as useful as considering the sume of "type of technology + type of Organism".

Thank you

Dear Participants,

I would like to kindly remind you that the forum will close tomorrow (Tuesday 25 April 2023) at 17:00 EDT. There is still time to share your valuable inputs, focusing on the types of engineered gene drives to consider in the development of additional voluntary guidance materials to support case-by-case risk assessment of LMOs containing engineered gene drives in accordance with Annex III of the Cartagena Protocol.

Best regards,
Luciana

My name is Martin Batic, and I am the head of the Biotechnology Section at the Ministry of Environment, Climate and Energy, which is responsible for the biosafety framework in Slovenia. I have a PhD in biotechnological sciences. I have many years of experience in the field of biosafety, biotechnology, and environmental risk assessment of LMOs and I am a national focal point for the Cartagena Protocol.

The voluntary guidance material should focus primarily on gene drive mosquitoes. Nevertheless, the voluntary guidance material should also be useful and relevant for the case-by-case risk assessment of a wide range of gene drive applications planned in this field in the future, e.g. for the control of invasive species and pests or the protection/conservation of endangered species, etc.

Best regards,
Martin

My name is Marja Ruohonen-Lehto, and I work as a Senior Adviser at the Finnish Environment Institute. I have a PhD in genetics and have worked on biosafety issues for more than 25 years. I have been a member of previous AHTEGs on risk assessment and risk management and have participated in the Cartagena COP-MOP negotiations since 2006.

I would like to support the following interventions, in particular: # 11580, # 11607, # 11619, # 11623 and # 11662. Many thanks colleagues, nothing to add at this point. This is very useful for our future work.

While I forgot in my first intervention - many, many thanks for Luciana for moderating the discussions.