Topic 3: Part B, sections 7 to 9

My name is Christoph Then, I am working for Testbiotech, a science based CSO in Germany (http://www.testbiotech.org).

As Testbiotech has shown in its previous input (https://www.testbiotech.org/node/3037), the case of Anopoheles gambiae is especially suited to deal with first experiences in risk assessment of EGD-LMO. There are some interesting lessons learned from the existing data that may be absent in other cases. 

In any case, in the planning of this specific risk assessment, specific attention will be needed to include all relevant aspects of uncertainties and unknowns. For the overall conclusions, it is not only decisive which data are available but also those which are missing. Therefore, already during the planning of the specific risk assessment, adequate tools have to be identified that allow to systematically address uncertainties and unknowns throughout the whole process of risk assessment.

My name is Anastasia Matthies, I have more than 10 years of experience in Biosafety and Risk Assessment of LMOs as scientific officer at the German Federal Office of Consumer Protection and Food Safety. Firstly, I wish to thank for the opportunity to contribute to the discussions in this online forum. Further I would like to thank Ms. Luciana Pimenta Ambrozevicius for moderating this forum, and all colleagues contributing to the discussion.

The structure of Part B should reflect considerations of Part A in order to demonstrate how the developed additional guidance material works by means of an example. Referring to my posts on Topics 2 and 3, Part A needs major revision to limit it to specific aspects for risk assessment regarding engineered gene drive organisms. Following, this revised structure should be applied in Part B on living modified anopheles containing engineered gene drives.

In PART B, section 9.1, “Biological differences between EGD-LM mosquito and non-modified strains should be added and this can form bullet 2.

Dear all,

My name is Eder Toppa, I've been working for the Brazilian Ministry of Agriculture and Livestock for the past decade and I am currently the Head of Biosafety Service as well as I am member of the National Biosafety Commission. I attended the COP / MOP meetings held in Montréal last December and I am following all the discussion about gene drive.

Line 106: "Part B, Section 9.1. Non-modified strains, comparator activities"
As stressed by Mr. Ernst Wimmer [#11811], all the comparators should be mentioned, such as: LMOs that do not contain the engineered gene drive; other disease vector control strategies - Wolbachia; insect sterile technique; biocontrol; use of pesticides etc. - and the consideration of resistance induction when those alternatives are used. Another one, is the no action alternative and the impacts on the population should be considered.

Best regards,
Eder Toppa

Hi my name is Eva Sirinathsinghji, I work as a Research Associate for Third World Network.

I would like to come back to the issue of target species definitions. I echo the points made in comment #11831 that a species that is not a disease vector, cannot be defined as a target species. Such a definition would represent a significant departure from how gene flow to non-target organisms has been assessed in the past, where gene flow has been a risk to be avoided e.g. the spread of a transgene from a crop to a wild relative. Considering gene flow to a non-vector species as an intended design feature risks creating an assessment process that fails to assess the potential biodiversity impacts on a species, while providing no benefit to the intended design aim of addressing disease transmission.   Any such change risks steering guidance towards a pathway of acceptability over risk assessment.

As  an added concern, I would like to raise attention to a recent study where a new species of Anopheles was discovered in 2019. This raises uncertainties regarding current knowledge of the extent of the species complex, gene flow barriers and potential unintended spread of transgenes in unexpected ways doi: 10.1038/s41598-019-49065-5. Moreover, as far as I am aware, though if anyone has any further details on the project then I would much appreciate any shared information on this, current gene drive projects are for example, only assessing the ecological role of Anopheles gambiae, not the wider species complex, to inform risk assessments. This raises a potential disconnect between which species are being assessed for adverse impacts of gene drive releases, versus which may be omitted from a risk assessment process if non-vector species become defined as a target species.

Thanks

I would like to offer some points of correction to post #11836.  First, gene flow is not a "risk."  It is an exposure pathway.  Gene flow is a natural phenomenon that occurs all the time.  Movement of transgenes through gene flow has to be assessed the same way movement of other genes would be assessed to determine if the results of gene flow will cause harm.  Second, the definition of "target" and "non-target" species depend entirely on the application being considered.  These are defined by the case specific assessment.  There is nothing preventing consideration of a non-vector species as a "target" species.  Regardless of whether it is considered a target or a non-target, the effects of a gene drive on species in the environment would be considered in any risk assessment.  Being categorized as a target species does not mean those species are "omitted from a risk assessment process."  This statement reflects a misunderstanding about the way risk assessments are conducted.  It is also worth pointing out that assumptions about "the intended design aim" are not appropriately dealt with in discussion of a guidance document when they can only be considered in a case specific risk assessment. 

Second, it is simply untrue to say that current gene drive projects are only addressing the ecological role of Anopheles gambiae.  From the earliest discussions of considering the use of gene drive for malaria control all discussions that I am aware of have considered the Anopheles gambiae complex.  It's also worth noting that at present, a biology document on the Anopheles gambiae mosquito including the species complex, is currently in progress under the OECD Working Party on the Harmonisation of Regulatory Oversight in Biotechnology to help inform future risk assessments.

Dear colleagues,

My name is Ricarda Steinbrecher, I am a biologist and molecular geneticist with long work experience on biosafety, risks and risk assessment of LMOs – and have introduced myself further under Topic 1.

Section B, lines 99-100: Concerning part B of the draft outline it appears that the focus and remit has been reduced and limited down to Anopheles mosquitoes for malaria control as compared to the remit given by COP-MOP decision 10/10, wish tasks the AHTEG to “develop additional voluntary guidance materials for conducting case-by-case risk assessments of living modified organisms containing engineered gene drives ….. A specific focus of this material should be engineered gene drive mosquitos taking into account …. “

It is not clear to me why and how this remit has been changed, in particular as there are three taxonomic groups of mosquitoes that are currently being researched and developed as ‘engineered gene drive mosquitoes’, and as these cover a broad application spectrum with the aim to reduce the transmission of vector-borne diseases in humans, mammals and birds, (including yellow fever, avian and human malaria).

As reported in the scientific literature mosquitoes other than Anopheles (A. gambiae, A. arabiensis, A. stephensi, A. funestus) are:
Aedes aegypti (see for example: Buchman et al. 2019; Buchman et al. 2020; Li, Yang, et al. 2020;  Verkuijl et al. 2020; Reid et al. 2022; Anderson et al. 2023) and
Culex quinquefasciatus (Anderson et al. 2019; Anderson et al. 2020; Li, Li, et al. 2020;   Harvey-Samuel et al. 2023)

It would be helpful and important to learn how and why the decision has been altered, as I believe the AHTEG will need to agree with this alteration or broaden the spectrum again. This would also affect lines 102, 104, 107 and 109.

B-7 (line 102): It is not clear to me why this is being set out as a “case study” of an Anopheles mosquito rather than a guidance for conducting risk assessment. My understanding is that the guidance would be for any EGD mosquito and not just for Anopheles, and even less for just one specific Anopheles as the current “an” suggests. Please keep line 102 open according to the mandate and have any potential adjustment be addressed by the AHTEG.

B-9.1 (line 106). According to ANNEX III the comparator for an LMO is the non-modified parental line. Whilst there have been other suggestions, including in this online forum [e.g. #11811 and #11832], they should not be added to the outline as this outline has to be in line with Annex III. A discussion on comparators will take place under 4.3, including development of criteria and the consequences of lack of comparator. This will then inform point 9.1.

REFERENCES:

Anderson et al. 2019. “CRISPR/Cas9 gene editing in the West Nile Virus vector, Culex quinquefasciatus Say.” PLoS One 14 (11):e0224857. doi: 10.1371/journal.pone.0224857.

Anderson et al. 2020; “Expanding the CRISPR Toolbox in Culicine Mosquitoes: In Vitro Validation of Pol III Promoters.” Acs Synthetic Biology 9 (3):678-681. doi: 10.1021/acssynbio.9b00436.

Anderson et al. 2023; Anderson et al. 2023. “Closing the gap to effective gene drive in Aedes aegypti by exploiting germline regulatory elements.” Nat Commun. 14(1):338. doi: 10.1038/s41467-023-36029-7.

Buchman et al. 2019. “Engineered resistance to Zika virus in transgenic Aedes aegypti expressing a polycistronic cluster of synthetic small RNAs.” Proc Natl Acad Sci U S A 116 (9):3656-3661. doi: 10.1073/pnas.1810771116.

Buchman et al. 2020; “Broad dengue neutralization in mosquitoes expressing an engineered antibody” [published correction appears in PLoS Pathog. 2020 Apr 29;16(4):e1008545]. PLoS Pathog. 2020;16(1):e1008103. Published 2020 Jan 16. doi:10.1371/journal.ppat.1008103

Harvey-Samuel et al. 2023. “CRISPR-based gene drives generate super-Mendelian inheritance in the disease vector Culex quinquefasciatus.” PRE-PUBLICATION, not peer reviewed as yet: bioRxiv 2023.06.12.544656; doi: https://doi.org/10.1101/2023.06.12.544656

Li, Li, et al. 2020; “Methods for the generation of heritable germline mutations in the disease vector Culex quinquefasciatus using clustered regularly interspaced short palindrome repeats-associated protein 9.” Insect Mol Biol 29 (2):214-220. doi: 10.1111/imb.12626.

Li, Yang, et al. 2020;   “Development of a confinable gene drive system in the human disease vector Aedes aegypti.” Elife 9. doi: 10.7554/eLife.51701.

Reid et al. 2022; Reid et al. 2022. “Assessing single-locus CRISPR/Cas9-based gene drive variants in the mosquito Aedes aegypti via single-generation crosses and modeling.” G3 (Bethesda)12(12):jkac280. doi: 10.1093/g3journal/jkac280.

Verkuijl et al. 2020; “A CRISPR endonuclease gene drive reveals two distinct mechanisms of inheritance bias.” bioRxiv:2020.12.15.421271. doi: 10.1101/2020.12.15.421271.

Hello. My name is ROUAMBA Wend-Rabo Mathurin, from Burkina Faso's National Biosafety Agency. I have about ten years' experience in the regulation of GMOs in Burkina Faso. Based on the moderator's questions, Sections 7 to 9, the definition must be extended to the complex, because the species in this complex are closely related, share genetic characteristics and have a high level of interbreeding potential. In the case of the mosquitoes of the Anopheles Gambia s. l. complex, studies show that they interbreed and produce viable eggs. The specific case of An. gambiae and An. Coluzzii show that they mate freely and produce viable, fertile male and female offspring.

I won't reintroduce myself, as I have posted previously.  But I will once again thank the Secretariat and the moderator for providing the opportunity to comment.

We have been asked to provide specific feedback on the outline proposed as a starting point for "guidance" on risk assessment of a living modified Anopheles containing an engineered gene drive for malaria control.  In reviewing the outline, parts 7-9 as instructed for this section of the forum, I am struck by a couple of features.  First, key features for determining whether the document will be useful, including any text describing the scope and objective of the document, are missing.  Without this, we are left to assign our own objectives and then imagine a future document that may or may not meet them.  Evaluating the outline is more of a Rorschach study of the online forum participants than a meaningful evaluation, and it mirrors a recurrent challenge for risk assessors.  It is impossible to assess a risk unless you have an understanding of what it is you are trying to protect.  As much as I would like to be positive, it is difficult to maintain much hope that a document that begins with no clear, agreed purpose will be successful.

The outline, as it exists now, is very incomplete.  It is not sufficient basis to begin the development of risk assessment guidance for EGD in Anopheles.

My name is Felicity Keiper and I am participating in this forum as a representative of the Global Industry Coalition (GIC). I have posted comments under Topics 1 and 2 suggesting substantial restructuring and editing of Part A (#11847, #11849, #11850). For Part B, we agree with the comments of #11829 questioning what the content/subject of a "case study" should be. We do not consider a case study to be necessary, since "Part A" should be focused on EGD-LMO mosquitoes.

Dear All,

I am a multidisciplinary researcher with a Ph.D. in Biomedical Sciences, a Ph.D. in Mathematics, and a Habilitation in Data Security, and was nominated by ENSSER. I am grateful to participate here and appreciate the many valuable comments.
As a trans-disciplinary researcher, I would like to raise some concerns that others do not have covered.

Apologies for the long post – which applies to the all the Q’s to some extent (Parts A AND B).

I would like to echo some of the messages before that cautioned against some of the statements made so far as if those were based on clear scientific/public consensus, as if the issue was sufficiently resolved. As stated in #11845, “It is impossible to assess a risk unless you have an understanding of what it is you are trying to protect.” While I appreciate the hard work that has gone into this, one facet that has totally gone missing is that of deliberate misuse and security. Intentional misuse is briefly mentioned in #11834, but must be more than one isolated aspect/potential consequence.

Deliberate misuse really ought to DEFINE risk assessment. Biological risk covers a spectrum encompassing naturally occurring, unintended, and deliberate risks (The Royal Society and the International Council for the Life Sciences, 2009). Deliberate misuse, however, is different from dual-use per se as it involves a given certainty that some actors intend to afflict harm. This concept has been extensively studied by Information Security disciplines. It has been recognized that as long as there is a potential for malicious exploitation, that such potential always will be maliciously exploited (not “if”, but “when”).

The analogous problem extends throughout the life-sciences, particularly via synthetic biology and the underlying convergence of technologies (computers, cyber-physical interfaces, cloud, networks, AI, etc). However, risk assessment involving EGD-LMOs may be unique, nonetheless, offering even greater potential for misuse, for various reasons:

• As highlighted in #11809, we are dealing with many “first time” aspects and established examples where previous models have failed (further extended for example in #11853, #11834). All these are a huge potential for zero-day exploits. In other words, since all of this is new, it’s easy to be misused.
• We are really dealing with a paradigm shift as to the type and scope of the intervention, involving genetic, molecular, ecological, societal, religious values, and many others. The adverse impact could be just as, or in some regards even much more far-reaching than a nuclear weapon. Such high-impact scenarios are great targets for those wishing to do harm.
• The issue of uncertainty/limits of modeling has been extensively discussed. Some argue that with gene-drives we don’t encounter foundationally new issues. The perspective of security – covering deliberate misuse – adds a different dimension altogether: it would be akin to inviting bad actors, simply because known and unknown uncertainties are not carefully monitored or mitigated.
• A high-risk scenario with potentially extremely far-reaching impact across various “scale” measures.

At present, the risk-assessment excludes deliberate forms of misuse. Unfortunately, there is no sense where all this carefully established technology could go awry from a security perspective – i.e., for deliberate misuse. These days, IT-disciplines would never say that any product has security gaps, and therefore, an entirely new one would not be different than others – and just release it. Instead, they heavily rely on security-by-design and –by-default principles. They would never roll out a new internet service without such practices and policies in place. Much of these, of course, are because of legal considerations also, because attacks and misuse continue to happen.
I strongly propose, therefore, that the entire risk-evaluation framework INCLUDES “security” (to informally cover deliberate misuse potential) rather than “only” safety (loosely, “only” considering what could go wrong without malicious involvement).

This needs to be considered in key terms and notions, including

• Protective goals, assessment/measurement endpoints (vs. how all this could go awry in a malicious setting)
• What does uncertainty and limits of modeling mean in an adversarial context
• Pathways of harm, risk hypotheses, receiving environment (weak spots and even policy guidelines may be deliberately targeted and circumvented)
• Adverse effects (note: deliberate misuse may aim to achieve different objectives than via “pathogenesis” or harm to humans)
• Hazard – and misuse – characterization
• Likelihood of events vs. malicious intent and goals
• Evaluation of consequences and how they are mitigated, and by whom (again, note malicious aims)
• Risk vs benefits – in an adversarial context, through misinformation, this entire notion could heavily be distorted (akin to the saying “the thief masquerading as a police officer”)
• Likelihood of adverse events (as opposed to intent, etc, see e.g. https://www.frontiersin.org/articles/10.3389/fbioe.2023.1209054/full)
• Legislation and enforcement (who shares what responsibilities to prevent misuse, etc)
• Acceptability of risk – in an adversarial context, do we even know what that is?
• Choice of comparators: I suggest to substantially extend this, to ALSO include COMPARABLE (bio)risk assessment analyses done in other disciplines and contexts (e.g. see all the work done on “cyberbiosecurity”)
• Detection, identification, persistence and monitoring (may be deliberately disguised or hidden)
• “Scale” and impact (geographically, economically, socially, etc) (propose to use the scaling framework developed by Jack Heinemann et al.)

Very recently, I did a study that highlighted how the entire risk spectrum – including deliberate misuse potentials – may radically re-shape our conception regarding the origin of SARS-CoV-2. Central to my analysis are (genetic) contaminants, limits/misinterpretations of molecular and biologic pathways, outdated models, convergence of technologies, and more (https://www.frontiersin.org/articles/10.3389/fbioe.2023.1209054/full). I suggest that many parallels can be drawn here.

References:

The Royal Society and the International Council for the Life Sciences (2009). New approaches to biological risk assessment. Avaliable At: https://royalsociety.org/topicspolicy/publications/2009/biological-risk/.

Heinemann, Jack A., et al. "Differentiated impacts of human interventions on nature: Scaling the conversation on regulation of gene technologies." Elem Sci Anth 9.1 (2021): 00086. https://doi.org/10.1525/elementa.2021.00086

Mueller, Siguna. "Recombination between coronaviruses and synthetic RNAs and biorisk implications motivated by a SARS-CoV-2 FCS origin controversy." Frontiers in Bioengineering and Biotechnology 11 (2023). https://doi.org/10.3389/fbioe.2023.1209054

Dear all,

I am Christophe Boëte and I have introduced myself in a previous post.
I agree with Dr Steinbrecher regarding the fact that a Part B focusing only on Anopheles mosquitoes is restrictive. This should be clearly explained given the fact that other mosquito species (vectors of infectious diseases) are currently researched as EGD mosquitoes or these other species should be included as other case studies in order to provide guidance not only for EGD Anopheles.

My name is Thato Mogapi from the Department of Forestry, Fisheries and the Environment in South Africa. 

In the case of defining the target organism, para c indicates that specific focus of this material should be engineered gene drive mosquitos taking into account the current experience with the organism. The guidance can focus on mosquito species that are known disease vectors, including Anopheles and Aedes species. In the introduction, the guidance document can reflect of species that are being targeted to introduce engineered gene drives, for example in the Africa region.