To facilitate a gathering of additional  information, participants are asked to consider the following points in relation to this topic:
1. Review the potential positive and negative impacts of this trend and issue in synthetic biology on each of the three objectives of the Convention.

2. What is the timeframe for release or potential impact of self-limiting insect systems? Please provide examples of specific applications (e.g., species, molecular mechanism).

3. What are the potential gaps or challenges for risk assessment, risk management and regulation for this topic in synthetic biology? Evaluate the availability of tools to detect, identify and monitor the organisms, components and products of synthetic biology.

4. Review the potential social, economic, cultural, ethical, political, human health and/other relevant impacts of this trend and issue. What are the relevant considerations for IPLCs, women and youth?

5. Is the trend and issue attempting to address specific problems, and if so, what are these problems and their underlying causes? How else could these problems or causes be addressed?

6. What lessons can be learned of similar tools, techniques or applications in other domains? How might those lessons from elsewhere be relevant or shed insight in assessing this topic in the context of the aims of the Convention on Biological Diversity?

7. Where are limits of knowledge with respect to this trend and issue? Are there any other considerations that would be important to raise?

Dear colleagues,

It is my pleasure to welcome you to the Open-ended Online Forum on Synthetic Biology. The forum will be open from 6 to 15 November at 17.00 EST. I will be moderating the discussion and will provide support should the need arise. Please also bear in mind the forum guidelines that you can find on the website.

Thank you in advance for your engagement and I am looking forward to productive discussions!

Kind regards,
Florian Rabitz

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. In my contribution, I refer to points 2., 3. and 7 of the questions raised by the moderator.

Genetically engineered self limiting systems are expected to spread genetic information within undomesticated populations, include constructs to suppress or disrupt populations of flies (Ant et al., 2012) or mosquitoes (Windbichler et al., 2008; Evans et al., 2019; Waltz, 2021) or agricultural pest insects such as fall armyworm and backdiamond moth (Reavy et al., 2022; Shelton et al., 2020) by introducing lethal gene constructs. Recent experimental releases were performed by company Oxitec in the US (mosquitoes and backdiamond moth) and Brazil (mosquitoes and fall armyworm). Further trials were conducted by Target Malaria in Burkina Faso  (Target Malaria, 2019).

There are several specific questions concerning risk assessment of these self limiting systems such as genetic stability, incomplete penetration (of the lethal function), the possibility to escape the lethal mechanisms due to environmental conditions, horizontal gene transfer, replacement by other species with potential harmful characteristics, problems in monitoring, unintended introduction of additional genetic material in target populations, inadequate buffer zones, uncontrolled large distance distribution due to heavy wind conditions, transboundary movements, potential impact on food webs, impact on non-target species and unintended consumption by humans (for references see for example: GeneWatch UK 2019 and 2021).

As the example of the Target Malaria release of Anopheles mosquitoes shows, several wrong or inadequate assumptions were made before the experimental release concerning the biology of the strain used for the trials, the potential for horizontal gene transfer and the site of the insertion of the additional gene construct. Such inadequate  assumptions may severely impact the safety of the Synbio mosquitoes after release (for references see Testbiotech, 2023).

The open questions in regard to risk assessment and the findings from trials of Target Malaria show a high level of complexity, going along with a broad range of uncertainties and unknowns that require a priority for the application of the precautionary principle, including the introduction of cut off criteria (Then et al 2020).

Ant T., Koukidou M., Rempoulakis P., Gong H.‐F., Economopoulos A., Vontas J., Alphey L. (2012) Control of the olive fruit fly using genetics‐enhanced sterile insect technique. BMC Biol 10(1):51. https://doi.org/10.1186/1741-7007-10-51

GeneWatch UK (2019) comments on docket identification (ID) number EPA-HQ-OPP-2019-0274-0001: New Active ingredient for Oxitec OX5034 Aedes aegypti mosquitoes, http://www.genewatch.org/uploads/f03c6d66a9b354535738483c1c3d49e4/GeneWatch_EPA_Oxitec_consul19_fin.pdf

GeneWatch UK (2021) response to the EPA’s consultation on an application 93167-EUP-2 from Oxitec (Docket No. EPA-HQ-OPP-2019-0274), http://genewatch.org/uploads/f03c6d66a9b354535738483c1c3d49e4/genewatch-uk-response-to-the-epa.pdf

Evans B.R., Kotsakiozi P., Costa-da-Silva A.L., Ioshino R.S., Garziera L., Pedrosa M.C., Malavasi A., Virginio J.F., Capurro M.L., Powell J.R. (2019) Transgenic Aedes aegypti mosquitoes transfer genes into a natural population. Sci Rep 9: 13047. https://doi.org/10.1038/s41598-019-49660-6

Reavey et al. (2022) BMC Biotechnology https://doi.org/10.1186/s12896-022-00735-9

Shelton AM, Long SJ, Walker AS, Bolton M, Collins HL, Revuelta L, Johnson LM and Morrison NI (2020) First Field Release of a Genetically Engineered, Self-Limiting Agricultural Pest Insect: Evaluating Its Potential for Future Crop Protection. Front. Bioeng. Biotechnol. 7:482. doi: 10.3389/fbioe.2019.00482

Target Malaria (2019) Target Malaria proceeded with a small-scale release of genetically modified sterile male mosquitoes in Bana, a village in Burkina Faso. https://targetmalaria.org/target-malaria-proceeded-with-a-small-scale-release-of-genetically-modified-sterile-male-mosquitoes-in-bana-a-village-in-burkina-faso/

Then, C. Kawall, K., Valenzuela, N. (2020) Spatio-temporal controllability and environmental risk assessment of genetically engineered gene drive organisms from the perspective of EU GMO Regulation. Integr Environ Assess Manag, 16(5), 555-568. https://doi.org/10.1002/ieam.4278

Waltz E. (2021) First genetically modified mosquitoes released in the United States. Nature 593(7858): 175-176. https://doi.org/10.1038/d41586-021-01186-6

Windbichler N., Papathanos P.A., Crisanti, A. (2008). Targeting the X chromosome during spermatogenesis induces Y chromosome transmission ratio distortion and early dominant embryo lethality in Anopheles gambiae. PLoS Genet 4(12), e1000291. https://doi.org/10.1371/journal.pgen.1000291

Dear colleagues, I’m Kathleen Lehmann, policy officer at the European Commission, Directorate-General of Health and Food Safety, biotech unit. I’m a biochemist by training. In the Commission I’m taking care of questions in synthetic biology and genetically modified (GM) microorganisms primarily in industrial biotech and pharmaceutical uses. I’ve several years experience with the risk assessment of GM microorganisms in contained use and the environmental risk assessment for the market authorisation of GM medicines. I’m a member of the mAHTEG.

I've a bit of a longer reply trying to go through all the questions raised.

On question 1:
• For vector control applications:
As relates to the objectives of the Convention potential negative impacts could arise in relation to biodiversity if an insect population collapses or is significantly diminished in response to the use of self-limiting insect systems. In this case also other species could be negatively affected through ecosystem interactions, for example changes in the food web. Additionally, other species might fill the vacant ecological niche, which might also impact ecosystems. Such effects need to be addressed in an appropriate environmental risk assessment.
Potential positive impacts do not relate to the objectives of the Convention and are addressed under question 4.

• For agricultural applications/pest management:
The intended positive impact is the protection of crop and livestock from insect pests or diseases transmitted through them, which would in turn increase the economic value in agriculture. As an application used in integrated pest management/population control these systems have the potential to reduce the negative impact of conventional agricultural practices based on the use of broad-spectrum pesticides/insecticides. They could therefore be considered under the objective of sustainable use of biological diversity or under conservation of biological diversity.
Potential negative impacts should a population collapse or significant decreases result from the use of the application are the same as for vector control applications and need to be addressed in an appropriate environmental risk assessment.

On question 2:
Release of genetically modified self-limiting insect systems has occurred at least under supervision of Oxitec in Brazil and the USA (sterile male Aedes aegyptii mosquitoes) and Target Malaria in Burkina Faso (male bias Anopheles gambiae mosquitoes).

On question 3:
• Before release, environmental data on population densities and dynamics of target and potentially non-target species, species interactions and dependencies as well as movement patterns are needed to inform the environmental risk assessment.
• After the release these parameters would need to be monitored to detect and evaluate any intended or unintended changes.
• At least for commercial applications the development of detection and identification methods can be assumed to be part of the commercial development.

On question 4:
• For vector control applications:
- In case of successful limitation or elimination of the spread of a pathogen,
a) reduction in cases of and deaths caused by vector-borne diseases (particularly in disproportionately affected children);
b) decreased health care costs and costs for other mitigation measures;
c) decreased loss of economic output due to illness and death;
d) increased economic security in affected communities;
e) area wide effect ensures protection independent of individuals’ characteristics such as wealth or education
- The decision to use or not use the application may or may not be based on a democratic process but will always affect all individuals of a community; an opt-out of individual members of the community will not be possible; this may challenge the concept of individual consent in health-related questions. However, this is also true for other vector control methods like use of insecticides.

• For agricultural applications/pest management:
- Reduced use of insecticides and thus of their harmful effects on non-target organisms and potentially human health;
- Positive economic impact due to reduction of output loss for agricultural products;
- The potential positive impacts on agricultural output will be to the benefit of a defined group of individuals, while potential negative consequences may affect all members of a community. This raises the general questions of fairness, liability and on who should be involved in the decision making on the use.

On question 5:
• For vector control applications:
Intends to reduce or disrupt transmission of pathogens with relevance for human health;
Direct alternatives are the use of bed nets, insecticides, gene drives, Wolbachia-mediated techniques
Indirect alternatives to mitigate the impact are the development and use of drugs and vaccines.

• For agricultural applications/pest management:
Intends to reduce or disrupt transmission of pathogens with relevance for animal or plant health or to reduce insect populations feeding on crop.
Direct alternatives are the use of insecticides, planting of insect resistant crops (genetically modified (GM) and non-GM), avoidance of monocultures

On question 7:
The designation as “self-limiting” insect system may also apply to certain gene drive applications, concretely systems that are designed to spread if present at high frequency while remaining spatially contained if present in low frequency (i.e. threshold-dependent drive systems) or systems that harbour parts of the drive in independent genetic loci which may be temporally confined if the parts are individually lost due to high fitness costs (e.g. in daisy chain drives) (see Marshall JM, Akbari OS. 2018. doi:10.1021/acschembio.7b00923). In view of the existence of topic 3, it would be beneficial for the final report to define that impacts of gene drives are not addressed under topic 2.

Best,
Kathleen

Dear colleagues,
My name is Dr. Swantje Schroll and I work at the German Federal Office of Consumer Protection and Food Safety as a scientific officer in the office of the German Central Committee for Biological Safety (ZKBS, https://www.zkbs-online.de/ZKBS/EN/Home/home_node.html), where I have been responsible for risk assessment of Synthetic Biology for the last ten years (see also post #3105).
Self-limiting insect systems have been explored for more than 20 years (see Thomas DD et. al. 2000. Science 287(5462): 2474-6) and can hardly be considered as a new trend. They use conventional methods of genetic engineering and the insects are considered as classical genetically engineered organisms, respectively LMOs, for which risk assessment strategies already exist. It is unclear why these systems need special analysis under the term “Synthetic Biology”.

Best regards,
Swantje

Dear Colleagues, thank you for this discussion and thank you very much to the moderators and chairs for this important discussion. I am Eva Sirinathsinghji, I am a biosafety research associate with Third World Network, and am a current and previous member of the RA AHTEG, and current member of this synbio mAHTEG.

I would first like to support the inputs on challenges to risk assessment raised by Christoph Then. I will take my opportunity to thus focus on other aspects, particularly with regard to SE, ethical and political considerations.

Q1.

Self-limited insects raise numerous concerns for the conservation of biodiversity, including for example:

-complex population responses e.g. high release numbers (and subsequent exposure of crops or people to pest/mosquito), population rebounds, movement of wild mosquitoes/pests (as appears to have happened during Oxitec trials in Cayman Islands (GeneWatch, 2021)
- exposure to LM males (or LM females that survive killing mechanism via resistance or due to survival at larval stage, unintended releases),
- use of non-native strains and generation of hybrid strains (e.g. Evans et al., 2019)
- use of antibiotics to feed insects
- Oxitec self-limited insects such as OX5034 insects include the release of male mosquitoes which will increase persistence in the environment in comparison to earlier versions of the technology
-exposure of humans and animals to biting female mosquitoes (see above for mechanism) with potential risks of toxicity/allergenicity
- response from competitor species e.g. niche replacement with alternative disease vector, or rise in secondary agricultural pest)
- genetic instability and next-generation effects in wild populations

see GeneWatch UK response to the EPA’s consultation on an application 93167-EUP-2 from Oxitec (Docket No. EPA-HQ-OPP-2019-0274) http://genewatch.org/uploads/f03c6d66a9b354535738483c1c3d49e4/genewatch-uk-response-to-the-epa.pdf


Q4,5,6:

There are potential impacts on loss of traditional knowledge e.g. resulting from the entrenchment of certain hegemonic trends/practices within global health and agriculture.

The technology raises concerns that it may provide benefits to particular stakeholders with potential negative impacts on others. For example, the use of self-limited LM fall armyworm is being suggested to only be suitable for agricultural systems using Bt crops alongside non-Bt refuge systems (Reavey et al., 2022 https://doi.org/10.1186/s12896-022-00735-9). Small-holder farming systems where refuges are not always implemented for a variety of reasons, are thus not likely set to even theoretically benefit from this technology, while being exposed to the potential risks.

A clearer understanding of who is to potentially gain or lose out from rolling out these technologies needs to be carefully addressed, as the details of the limitations are not always clearly presented, especially in information targeted more widely at the public.

Potential negative social, economic, health and political impacts are raised by potential efficacy failures of self-limited insect technologies. Efficacy failures raise economic concerns for the cost of such technologies as well as the opportunity costs that may arise from neglecting alternative, more sustainable solutions.

As raised above with regard to agricultural pests such as the LMO fall armyworm, developers suggest that this product would not work without being applied to Bt crop fields that implement a refuge strategy, and would require regular, sustained releases of the GE insect. Moreover, this product has been trialled in the field yet there was no empirical data provided to show any reduction in pest damage (Reavey et al., 2022 https://doi.org/10.1186/s12896-022-00735-9).

The required use of Bt crops (with at least two Bt toxins) with refuges, alongside the LM fall army worm also raises questions regarding the wider LMO approach to dealing with fall armyworm. Fall armyworm has rapidly developed resistance to Bt crops with documented resistance to all but a single Bt toxin remaining.  With the utility of Bt crops now coming into question for fall armyworm (amongst other pests), this technology may serve little more than to hold up and prolong the use of a declining product for a few more years (for a summary and references on the declining efficacy of Bt crops, resistance issues with fall army worm and implementation of refuges, please see our recent briefing https://www.twn.my/title2/biosafety/pdf/bio19.pdf). Of more concern, is the rolling out a technology that requires sustained releases, potentially generating dependence and technological lock-in not just to Bt crops, but now also to Bt insects, which would all need to work in conjunction.

With regard to mosquitoes, the issue of efficacy is also crucial. Data is lacking to show evidence of addressing the intended goal of suppressing disease. For example, with regard to Oxitec’s first generation mosquito, freedom of information requests revealed a lack of data to show reduction in mosquito numbers, despite public statements made to the contrary. As reported by GeneWatch, 2019:
“Oxitec’s claims of past success have been highly misleading. For example, Freedom of Information requests to the Cayman Islands’ Mosquito Research and Control Unit revealed comments from scientists with access to the data such as:
• “Whilst Oxitec and MRCU are making public statements proclaiming major reductions in the Aedes aegypti population in the treatment area the data I have seen does not support this.”
• “To date all the measures recorded have shown no significant reduction in the abundance of Aedes aegypti in the release area.”
The WHO Vector Control Advisory Committee have also raised this issue with regard to OX513A mosquitoes claiming that results from epidemiological trials remain the primary missing information for assessment of the public health value of this product. Epidemiological studies must be carried out to assess the public health value of reducing vector populations through the application of OX513A (WHO 2017: Seventh meeting of the vector control advisory group (VCAG). World Health Organization, Department of Control of Neglected Tropical Diseases, Geneva, Switzerland. Available at: https://tinyurl.com/yxhzdsxc) (see also Boete, 2021 DOI 10.3920/978-90-8686-895-7_12). The WHO report also stated that more evidence of sustainability of the product was needed.
This issue is still yet to be addressed in novel trials with the second generation OX5034 mosquito however.  Recent trials in Florida were not aimed at assessing reduction in disease, as reported further by GeneWatch (2019):
“The EPA states, ““...because the OX5034 mosquitoes are intended for suppression of Ae. aegypti mosquito populations and are not intended to directly influence disease transmission, epidemiological studies assessing effects on disease transmission are not required...” (p.106, EPA-HQ-OPP-2019-0274-0355). However, there is a complex relationship between mosquito numbers and disease transmission and evidence of population suppression is not sufficient to show a reduction in disease, or risk of disease.”
Unless I am not up to date on information and would be happy for any data to be shared here, I am not aware of any data provided by Oxitec that their mosquitoes have had any impact on disease suppression, yet claims are made, including by funders, to the contrary.
In my view, interrogation of claims and an assessment of the validity of claims is a vital part of this assessment process if challenges of vector-borne disease and other agricultural issues are to be successfully addressed.


GeneWatch (2019): http://genewatch.org/uploads/f03c6d66a9b354535738483c1c3d49e4/genewatch-uk-response-to-the-epa.pdf

In my view, taking into consideration the lack of evidence of efficacy of self-limited insects to date, we must be extremely careful not to 1. Rely on developer claims, 2. conflate the WHO’s investigation into the potential use of a novel product, with evidence of efficacy.
Considering claims of benefits and evidence of efficacy carefully is warranted so as to not divert attention and investment away from current control programs, or undermine frontline responses. As raised in Boete and Reeves (2018 DOI: 10.1016/S2214-109X(16)00084-X): “Use of insecticides and destruction of mosquito breeding sites had a central role in simultaneously eliminating Aedes aegypti from 18 continental countries, including Brazil, between 1947 and 1962.5 Effectively fighting mosquitoes and the diseases they transmit has generally required community participation in the application of sustainable and cost- effective approaches.”
Moreover, a narrow focus on vector control may risk marginalizing key health determinants such as strengthening healthcare systems, access to treatments, poverty alleviation and wider sanitation interventions, which should be incorporated into the technology assessment discussions. Biomedical reductionism through an over focus on technological interventions has been linked to foreign priorities of global health agendas, that are often driven by the Global North, and moreover, linked to international health, economic and security policy.

Ethical issues surrounding consent are also relevant to self-limited insects, including those used by gene-drive developers (thus relevant to the topic of gene drives in the other section of the forum). For example, civil society organisations have noted issues surrounding the lack of consent for the release of LM mosquitoes in Burkina Faso: https://www.etcgroup.org/content/target-malarias-gene-drive-project-fails-inform-local-communities-risks-new-film

With regard to the US Oxitec trials, GeneWatch also report that: “Oxitec is not required under EPA’s human studies rule to obtain informed consent of those living in the areas where the Oxitec mosquitoes would be released...” (EPA-HQ-OPP-2019-0274, p. 137). This is based on the finding that, “...the research involved with Oxitec’s release of mosquitoes does not meet the regulatory definition of research involving human subjects. Because the proposed information to be collected as part of this research does not involve human subjects, it is not necessary to evaluate whether the research would constitute intentional exposure of human subjects”.
They further state:
“However, human subjects will be living in the release areas. They may be bitten by surviving adult female GE mosquitoes, swallow male or female GE mosquitoes, and their risk of mosquito-borne diseases could increase if wild mosquitoes move to a different area, or there is a rebound of Aedes aegypti mosquitoes following the releases, or if the competitor species Aedes albopictus moves into the area. It is illogical to require consent only if these potential adverse impacts on humans are monitored and assessed.
The EPA’s view that consent is not required is therefore ethically and legally extremely questionable.”

Thanks very much and I look forward to contributing to the deep dive assessment on this issue in the meeting next February.
Eva

Dear colleagues, I’m Luke Alphey, Professor of Genetics at the University of York, UK. I have >20 years experience developing synbio insects.

Kathleen Lehmann raises an important point (#3101, while addressing Q7, though this may apply to other questions as well) in that designs for self-limiting gene drives are well established in the literature. This goes rather beyond the description of self-limiting insect systems in the briefing note, which relates to improvements on the radiation-based sterile insect technique. Self-limiting may be defined [1] as relating to temporal dynamics, so that the modification will not pass on indefinitely through subsequent generations, even in the absence of mutation or heritable resistance.  For gene drives, this will typically mean that the allele frequency of the gene drive will initially increase (the gene drive phenomenon) but later decrease, eventually disappearing. As noted in #3101 this includes systems that harbour non-driving components that decline in allele frequency due to fitness cost, while the drive properties of other elements depend on the non-driving components. Examples include Killer-Rescue [2], and various “split” drives, e.g. split homing drives such as daisy drives [3], or split-ClvR [4], for example. #3101 also refers to high-threshold drives. These may be “local”, i.e. they are not expected to spread substantially beyond the target area or population [1], but not necessarily self-limiting in the temporally-restricted sense above. For example, underdominance-based drives [5]. Either self-limiting or local drives (and most self-limiting drives are also local) may mitigate many of the concerns raised regarding more invasive/persistent gene drives. A key question in risk assessment for such drives would be whether there is any plausible route for the drive in question to lose its self-limiting characteristic, i.e. to mutate to a self-sustaining version. This will depend strongly on the specifics of the particular molecular-genetic design.

Q2 as alluded to by both #3101 and #3089, self-limiting insect systems have been in field use for well over a decade, in several countries and on substantial scales (Oxitec successfully released ca 1 billion Aedes aegypti OX513A males before switching to the more advanced OX5034 strain). OX5034 is female-lethal, so the transgene persists in the field beyond the release generation, but is rapidly eliminated due to high fitness cost (female-lethal) in the absence of further releases. In this regard it is self-limiting but not ‘immediately’ eliminated. No self-limiting gene drive systems – which are expected to persist longer, perhaps temporarily increasing in allele frequency before ultimately declining in frequency in the absence of further releases – have yet been released. In relation to another self-limiting release, that of modified Anopheles gambiae by Target Malaria, the assessment of #3050 seems rather harsh. The key feature that makes that the mosquitoes in question (Ag(PMB)1) self-limiting is the autosomal location. That was well established by simple genetics. That the precise insertion site was not where initially reported does not change the risk profile – indeed the publication of that correction in the scientific literature seems a model of transparency. Furthermore, I do not think that was the strain used in the 2019 release, which was Ac(DSM)2 [6]. In addition to the published literature, Target Malaria have a number of helpful factsheets on their website for anyone who might be confused.

With only the small caveat above regarding self-limiting gene drive systems, I would agree with #3106 that “Self-limiting insect systems have been explored for more than 20 years (see Thomas DD et. al. 2000. Science 287(5462): 2474-6) and can hardly be considered as a new trend. They use conventional methods of genetic engineering and the insects are considered as classical genetically engineered organisms, respectively LMOs, for which risk assessment strategies already exist. It is unclear why these systems need special analysis under the term “Synthetic Biology”.

1. Alphey LS, Crisanti A, Randazzo F, Akbari OS: Standardizing the definition of gene drive. Proceedings of the National Academy of Sciences 2020, 117(49):30864-30867.
2. Gould F, Huang Y, Legros M, Lloyd AL: A Killer–Rescue system for self-limiting gene drive of anti-pathogen constructs. Proceedings of the Royal Society B: Biological Sciences 2008, 275(1653):2823-2829.
3. Noble C, Min J, Olejarz J, Buchthal J, Chavez A, Smidler AL, DeBenedictis EA, Church GM, Nowak MA, Esvelt KM: Daisy-chain gene drives for the alteration of local populations. Proceedings of the National Academy of Science (USA) 2019, 116(17):8275-8282.
4. Oberhofer G, Ivy T, Hay BA: Split versions of Cleave and Rescue selfish genetic elements for measured self limiting gene drive. PLOS Genetics 2021, 17(2):e1009385.
5. Davis S, Bax N, Grewe P: Engineered underdominance allows efficient and economical introgression of traits into pest populations. Journal of Theoretical Biology 2001, 212(1):83-98.
6. Yao FA, Millogo AA, Epopa PS, North A, Noulin F, Dao K, Drabo M, Guissou C, Kekele S, Namountougou M et al: Mark-release-recapture experiment in Burkina Faso demonstrates reduced fitness and dispersal of genetically-modified sterile malaria mosquitoes. Nat Commun 2022, 13(1):796.

Dear Colleagues,

an important concern regarding self-limited categorisations for gene drives has been raised in the RA discussions, but perhaps are also relevant here with regard to determining safety and scope of different types of self limited drive systems. It is my view that definitions of these 'self limited' systems 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

Many thanks,
Eva

Dear participants,

We are Váleri Vásquez, a postdoctoral fellow at Stanford University in the Center for International Security and Cooperation as well as the Department of Biology, Omar Akbari, Professor, and Robyn Raban, Research Project Manager from the University of California Department of Cell and Development. Valeri’s research includes the computational modeling of transgenic mosquitoes for biocontrol and Omar and Robyn focus on mosquito biology, genetics, and genome engineering. We appreciate the opportunity to engage with this discussion hosted by the Secretariat and here contribute to points 1, 2, 3, 6, and 7 under Topic 2, “self-limiting insect systems.”

Self-limiting insect systems, designed such that they are not sustained in the wild population absent continued releases, have been historically successful pest management tools. These non-gene drive technologies include sterile insect technique (SIT) in the agricultural space as well as the release of insects with dominant lethal (RIDL) and incompatible insect technique (IIT) for public health purposes. A comprehensive review of genetic biocontrol is furnished by Raban et al (2023).

Regarding Point 1: Self-limiting insect systems are supportive of the objectives of the convention in that they are species-specific, upholding the conservation of biological diversity by directly targeting the pest of concern. Self-limiting insect systems also enable the fair and equitable sharing of benefits arising out of the utilization of genetic resources: to date, there are numerous agricultural and public health successes recorded around the world. Examples of the suppression of mosquito disease vectors using self-limiting insect systems include trials in the Cayman Islands, Brazil, the United States, and China (Crawford et al. (2020), Zheng et al. (2019), Mains et al. (2016), Carvalho et al. (2015), Harris et al. (2011, 2012), Lowe et al. (1980)).

Regarding Point 2: In addition to the historical examples noted above, a new technology that uses CRISPR to generate sterile males, pgSIT, is described in Raban et al (2023). Specifics on the timeframe for release and potential impact of pgSIT releases on malaria transmission and local economics in a malaria endemic region of The Gambia are discussed in Gendron et al (2023). 

Regarding Point 3: Several means of monitoring self-limiting insect populations have been employed historically via trapping methods targeting both the adult and aquatic stages (e.g., of mosquito vectors) to enable surveillance of the male-to-female ratio, the sterile-to-wild male ratio, and fluorescent markers (Harris et al., 2012). Genetic methods not yet deployed in the wild might also employ protein or genetic markers, and monitoring of such systems is recommended to continue beyond the active study period (James et al., 2018).

Regarding point 6: Many self-limiting insect systems build upon the gold standard insecticide-free insect control technology, sterile insect technique (SIT).  The SIT technology relies on the release of sterilized males en masse to suppress pest populations and has been used for decades to successfully control agricultural pests.    A brief history of genetic biocontrol and SIT can be found in Raban et al 2023. This review also describes a new technology, termed pgSIT which uses CRISPR genome engineering to generate precise mutations in genes to generate sterile males.  This technology was first built and described in the model fly, Drosophila melanogaster (Kandul et al 2019 and Kandul et al 2021) and has since been built in agricultural pests (Kandul et al 2022), the dengue, yellow fever, Zika and chikungunya vector mosquito, Aedes aegypti (Li et al 2021, 2023), and the malaria vector mosquito, Anopheles gambiae (Smidler et al 2023). 

Regarding point 7:  Self-limiting technologies, such as traditional SIT, have been studied for decades in the laboratory and field, so much is known about their behavior and impact on non-target species and the environment.  Newer self-limiting technologies have not been vetted in the long term, but by their nature would quickly be removed from the population when releases are discontinued. Should negative impacts appear in field trials, their limited scope and the transient nature of the technology would likewise limit the spatial and temporal impact of the technology. 

Thank you, we look forward to the continuing conversation on this platform.


References:

Li, M., Kandul, N. P., Sun, R., Yang, T., Benetta, E. D., Brogan, D. J., ... & Akbari, O. S. (2023). Targeting Sex Determination to Suppress Mosquito Populations. doi: 10.1101/2023.04.18.537404

Liu, X., Goldsmith, C. L., Kang, K. E., Vedlitz, A., Adelman, Z. N., Buchman, L. W., ... & Medina, R. F. (2023). General science‐technology orientation, specific benefit–risk assessment frame, and public acceptance of gene drive biotechnology. Risk Analysis. doi: 10.1111/risa.14242

Raban, R., Marshall, J. M., Hay, B. A., & Akbari, O. S. (2023). Manipulating the Destiny of Wild Populations Using CRISPR. Annual Review of Genetics,
doi: 10.1146/annurev-genet-031623-105059.

Smidler, A. L., Apte, R. A., Pai, J. J., Chow, M. L., Chen, S., Mondal, A., ... & Akbari, O. S. (2023). Eliminating malaria vectors with precision guided sterile males. bioRxiv.

Kandul, N. P., Liu, J., Buchman, A., Shriner, I. C., Corder, R. M., Warsinger-Pepe, N., ... & Akbari, O. S. (2022). Precision guided Sterile males suppress populations of an invasive crop pest. GEN Biotechnology, 1(4), 372-385.

Gendron, W., Raban, R., Mondal, A., Sánchez C., H.M., Smidler, A., Zilberman, D., Ilboudo, P.G., D’Alessandro, U.,  Marshall, J.M., Akbari, O.S. Cost-effectiveness of Precision Guided SIT for Control of Anopheles gambiae in the Upper River Region, The Gambia. bioRxiv 2023.07.20.549762; doi:https://doi.org/10.1101/2023.07.20.549762

Marshall, J. M., & Vásquez, V. N. (2021). Field trials of gene drive mosquitoes: lessons from releases of genetically sterile males and Wolbachia-infected mosquitoes. Genetically Modified and other Innovative Vector Control Technologies: Eco-bio-social Considerations for Safe Application, 21-41.

Kapranas, A., Collatz, J., Michaelakis, A., & Milonas, P. (2022). Review of the role of sterile insect technique within biologically‐based pest control–An appraisal of existing regulatory frameworks. Entomologia Experimentalis et Applicata, 170(5), 385-393.

Kandul, N. P., Liu, J., & Akbari, O. S. (2021). Temperature-inducible precision-guided sterile insect technique. The CRISPR Journal, 4(6), 822-835.

Li, M., Yang, T., Bui, M., Gamez, S., Wise, T., Kandul, N. P., ... & Akbari, O. S. (2021). Suppressing mosquito populations with precision guided sterile males. Nature Communications, 12(1), 5374.

Kandul, N. P., Liu, J., Sanchez C, H. M., Wu, S. L., Marshall, J. M., & Akbari, O. S. (2019). Transforming insect population control with precision guided sterile males with demonstration in flies. Nature communications, 10(1), 84.

Crawford JE, Clarke DW, Criswell V, Desnoyer M, Cornel D et al (2020) Efficient production of male Wolbachia-infected Aedes aegypti mosquitoes enables large-scale suppression of wild mosquitoes. Nat Biotechnol 38:482–492

Zheng X, Zhang D, Li Y, Yang C, Wu Y et al (2019) Incompatible and sterile insect techniques combined eliminate mosquitoes. Nature 572:56–61

James, S., Collins, F. H., Welkhoff, P. A., Emerson, C., Godfray, H. C. J., Gottlieb, M., ... & Touré, Y. T. (2018). Pathway to deployment of gene drive mosquitoes as a potential biocontrol tool for elimination of malaria in sub-Saharan Africa: recommendations of a scientific working group. The American journal of tropical medicine and hygiene, 98(6 Suppl), 1.

Mains JW, Brelsfoard CL, Rose RI, Dobson SL (2016) Female adult Aedes albopictus suppression by Wolbachia-infected male mosquitoes. Sci Rep 6:33846

Carvalho DO, McKemey AR, Garziera L, Lacroix R, Donnelly CA et al (2015) Suppression of a field population of Aedes aegypti in Brazil by sustained release of transgenic male mosquitoes. PLoS Negl Trop Dis 9:e0003864

Harris AF, McKemey AR, Nimmo DD, Curtis Z, Black I et al (2012) Successful suppression of a field mosquito population by sustained release of engineered male mosquitoes. Nat Biotechnol 30:828–830

Harris AF, Nimmo DD, McKemey AR, Kelly N, Scaife S et al (2011) Field performance of engineered male mosquitoes. Nat Biotechnol 29:1034–1037

Lowe RE, Bailey DL, Dame DA, Savage KE, Kaiser PE (1980) Efficiency of techniques for the mass release of sterile male Anopheles albimanus. Am J Trop Med Hyg 29:695–703

Dear Participants of the Open-ended Online Forum on Synthetic Biology,

Thank you for your interventions and active engagement.
The forum is now closed for comments.

Thank you,
The Secretariat

Re:2: Self-limiting insect systems

I agree with Luke Alphey that the description for “self-limiting” in the briefing issue describes only one approach for self-limiting insects. Self-limiting insects include those that produce sterile males but also those that for example produce fertile offspring with overrepresentation of one of the two sexes that may lead to reduced reproductive potential in the subsequent population (e.g. autosomal sex distorters have been developed for example in Anopheles gambiae (Galizi et al., 2014) and Ceratitis capitata (Meccariello et al., 2021).
Once releases stop these self-limiting insects will disappear within a limited number of generations from the population. Releases of self-limiting insects were approved already several years ago in the disease vectors Aedes aegypti (FriendlyTM developed by Oxitec) and in Anopheles gambiae (developed by Target Malaria, using the sterile male strain Ac(DSM)2 as pointed out correctly by Luke Alphey).
Thus, I agree with Swantje Schroll, self-limiting insects are not new and already captured under regulatory guidelines and frameworks for GMOs (WHO, 2021; Cartagena Protocol).

Thank you very much,
Silke

Publications include:
Guidance framework for testing genetically modified mosquitoes, second edition. Geneva: World Health Organization; 2021. Licence: CC BY-NC-SA 3.0 IGO
The Cartagena Protocol on Biosafety to the Convention on Biological Diversity defines a so-called living modified organism (LMO) in Article 3 (g) as “any living organism that possesses a novel combination of genetic material obtained through the use of modern biotechnology.” Modern biotechnology is further defined in Article 3 (i) as “the application of: a. In vitro nucleic acid techniques, including recombinant deoxyribonucleic acid (DNA) and direct injection of nucleic acid into cells or organelles, or b. Fusion of cells beyond the taxonomic family, that overcome natural physiological reproductive or recombination barriers and that are not techniques used in traditional breeding and selection.”
Meccariello, A., Krsticevic, F., Colonna, R. et al. Engineered sex ratio distortion by X-shredding in the global agricultural pest Ceratitis capitata. BMC Biol 19, 78 (2021). https://doi.org/10.1186/s12915-021-01010-7
Galizi, R., Doyle, L., Menichelli, M. et al. A synthetic sex ratio distortion system for the control of the human malaria mosquito. Nat Commun 5, 3977 (2014). https://doi.org/10.1038/ncomms4977