Category Archives: gene drive

National Academies of Sciences briefing on gene drive technology

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Today the NAS released a report on gene drive technology:

http://nas-sites.org/gene-drives/2016/05/26/report-release/

Excerpts from a New York Times article about the report:

'On Wednesday, the National Academies of Sciences, ... endorsed continued research on the technology, concluding after nearly a yearlong study that while it poses risks, its possible benefits make it crucial to pursue. ... The report underscores that there is not yet enough evidence about the unintended consequences of gene drives to justify the release of an organism that has been engineered to carry one. ... At the same time, it is uncertain how the technology will be regulated. Existing laws, the report noted, are aimed at containing genetically engineered organisms rather than managing those whose purpose is precisely to spread swiftly. ... Coming up with an international regulatory framework is especially crucial, members of the committee said, given that gene drives will not recognize national or political boundaries. For now, the United States Food and Drug Administration has authority over animals that have been engineered with foreign DNA under a rule that regards them as a type of drug. But the report suggests that other agencies, like the Fish and Wildlife Service or the Bureau of Land Management, might be seen to have a stake in the ecological concerns at the heart of gene drive experiments. ... Some independent scientists say the panel, which included ethicists, biologists and others, struck a good balance by permitting more gene drive research while limiting the use of the technology. But opponents of genetic engineering argue that the panel should have demanded a halt to research on gene drives, at least until some of the many questions it raised are answered. ... The committee considered six case studies, including using gene drive to control mice destroying biodiversity on islands, mosquitoes infecting native Hawaiian birds with malaria, and a weed called Palmer amaranth that has become resistant to herbicides and a scourge for some farmers. Each potential use of gene drive carries its own set of risks and benefits, the report says, and should be assessed independently. ... The group recommends “phased testing,’’ which would include safeguards at each step before eventually releasing organisms into the wild, but it also noted the new ethical challenges posed by how to obtain consent from people whose environments might be affected by such a release. “There are few avenues for such participation,” the report noted, “and insufficient guidance on how communities can and should take part.”'

Safely Testing Gene-Drive Systems: Discouraging Responsibility in Science

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My recent news about being promoted with tenure has emboldened me to write about some things that I have kept pent up for a long time. I'm not sure if this is a good thing but lets see where this leads us.

We engineered and demonstrated a self-limiting gene-drive for local and reversible genetic modification of a population. There is an argument about whether this type of system, underdominance, should even be considered gene-drive in the broader sense---but I am not going to go into definition arguments here. It is certainly a much safer type of population transformation system than alternatives that can invade a population from arbitrarily low frequencies.

Dr. R. Guy Reeves and I first began discussing engineered haploinsufficient mediated underdominance in 2006 when I returned from some work in Africa. I hired him as a postdoc in my new position at the Max Planck Institute for Evolutionary Biology in 2008 and we began work engineering the system. We knew it would take years to troubleshoot the technology so we also published theoretical results to lay the groundwork for making predictions of underdominant systems, e.g. Altrock, P. M., Traulsen, A., Reeves, R. G., & Reed, F. A. (2010). Using underdominance to bi-stably transform local populations. Journal of Theoretical Biology, 267(1), 62–75. doi:10.1016/j.jtbi.2010.08.004

We made three inserts into the genome of Drosophila melanogaster of our engineered genetic construct and started testing them. How do you test if you have generated underdominace?  This requires tracking the frequency of the insert over multiple generations in multiple replicate populations, which takes time and is quite a bit of work. I spent many nights in the lab counting thousands of flies and then walking home for a couple hours of sleep before sunrise. The first insert was homozygous lethal and useless for engineering underdominance. The second insert had lower homozygous fitness than as a heterozygote (technically a hemizygote) and did not result in underdominance. The third insert was interesting. As I collected data and each generation went by it began to look more and more like underdominance and became more and more statistically significant. I remember late one day in the winter of 2009/2010 Guy was going to leave to take the train back home to Hamburg and I asked him to stay and catch the next train. I had a notebook full of new data and I wanted him to see how it came out when I plotted it and did the calculations. It was clear unambiguous underdominance! I presented the results at our next meeting in May 2010 (Reed, F. A. and R. G. Reeves. Underdominance theory meeting data, how do they get along? Aquavit VIII meeting, The Max Planck Institute for Evolutionary Biology, Plön, Germany. slides PDF link). I also presented the results at some other talks such as February 2011 in Hawai'i (slides PDF link). Obviously we were excited about this, wrote up our results, and submitted them for publication!

All along we were aware of the potential for unintended dynamics of the system; I mentioned just this concern in a publication in 2007 (Reed, F. A. (2007). Two-locus epistasis with sexually antagonistic selection: A genetic Parrondo’s paradox. Genetics, 176(3), 1923–1929. doi:10.1534/genetics.106.069997). We were aware of invasive Medea gene-drive dynamics and discussed the possibility of (unintended) maternal deposition of the RNA "poison" into embryos with rescue depending on transcription in the embryos---this could completely change the dynamics of the system. So, we built a fail-safe into our system. We divided expression control of the RNA "poison" over two chromosomes, that have to be together in the same individual for it to work, using a standard binary control system (GAL4/UAS). If genetically modified "gene-drive" flies escaped from the lab then independent assortment (male recombination is suppressed) of the chromosomes in the following generation would break the system and it would not be able to drive. This enabled safe testing in the lab and the binary control system was not required for actual future applications of the technology where the fail-safe could be removed from the system (we went to great pains to explain this to reviewers, backed up with facts about how our flies were transformed).

So, it turns out that we were unknowingly in competition with another lab to be the first to publish a self-limiting gene drive system. When we submitted our manuscript for publication we encountered a very hostile reviewer. (There were also other issues at play that delayed the process, the Max Planck system decided to pursue a patent on the technology, there were personalities involved, etc. However, the reviews were truly maddening and am what I am focusing on here.) This person tried to find a reason to reject the manuscript and focused on the fail-safe---claiming that our approach could not work without this in place. We were eventually rejected from publishing in journal after journal, and the hostile reviews followed us from journal to journal, in some cases with the exact same review copied from the previous journal submission despite our revisions to the manuscript. This dragged out over a period of years and then we were finally able to publish in PLoS ONE (Guy Reeves, R., Bryk, J., Altrock, P. M., Denton, J. A., & Reed, F. A. (2014). First steps towards underdominant genetic transformation of insect populations. PLoS ONE, 9(5). doi:10.1371/journal.pone.0097557). However, Akbari et al. was published in 2013 (Akbari, O. S., Matzen, K. D., Marshall, J. M., Huang, H., Ward, C. M., & Hay, B. A. (2013). A Synthetic Gene Drive System for Local , Reversible Modification and Suppression of Insect Populations. Current Biology, 23(8), 671–677. doi:10.1016/j.cub.2013.02.059).  Even though their names appear here, this is not an attack directed at Akbari, Hay, or anyone else named here; I do not know the names of the reviewers of our manuscript (and later our grant applications) and I am not implying that it is any of these people in particular.

Our system has a lot of potential advantages, not least of which is the likely species portability of the approach due to the ubiquity of haploinsufficiency of ribosomal proteins across species. The Akbari et al. 2013 approach depends on careful control of expression timing during development, and while it certainly could be ported across species this is likely to be more difficult. However, we seem to have been blackballed. When applying for grant funding to implement this system in mosquitoes I get comments back the reflect some of the hostile reviews we received earlier (that this system is "fanciful" and cannot work in the wild, etc.). I have seen presentations where Akbari et al. 2013 is credited with the first self-limiting gene drive (no, we presented our results in 2010, 2011, we have a patent priority date of 2012). And I see reviews where our system is described as only proof-of-principle while the Akbari et al. 2013 system is described in contrast as a "fully functional system capable of invading wild populations" (Champer, J., Buchman, A., & Akbari, O. S. (2016). Cheating evolution: engineering gene drives to manipulate the fate of wild populations. Nature Reviews Genetics, 17(3), 146–159. doi:10.1038/nrg.2015.34) ... wild populations of what, Drosophila melanogaster? Transforming D. melanogaster is not useful, being able to transform other species, such as mosquitoes, is what is useful. Furthermore, accidentally transforming the entire Drosophila melanogaster species is dangerous, for reasons that not least of which it is a useful model organism, a human commensal, and because of the potential public backlash this could cause.

In another publication that was also delayed for years by a hostile reviewer, perhaps even the same person, we recommended combining underdomiannce with gene-drive systems like Medea in order to protect laboratory model organisms from unintended species-wide genetic modifications (Gokhale et al. 2014, http://bmcevolbiol.biomedcentral.com/articles/10.1186/1471-2148-14-98). The point I am trying to make is that being thoughtful and safely designing gene-drive systems, with safety checks and fail-safes in place, should be encouraged rather than discouraged within the scientific community. Unfortunately, in my experience the opposite seems to be true.

Genetic Ice-nine ?

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I was talking to a group of undergraduates last week and came up with an analogy to one type of gene-drive system. Different forms of water exist, on Earth's surface we are familiar with the common solid, liquid, and gas forms. However, under different temperatures and pressure solid water, or ice, can fall into alternative crystal arrangements. There are 16 known forms of ice. We are used to ice-I, but there is also an ice-II, ice-III, etc. Some forms of ice (ice-VII for example, can exist at very high temperatures, above the boiling point of water, but only at very high pressures which forces the molecules together into a crystal structure).

Phase_diagram_of_water.svg

In Kurt Vonnegut's novel Cat's Cradle ice-nine is a crystal form of water, based loosely on the idea of alternative forms of ice. In the book ice-nine freezes at room temperature, importantly it also causes liquid water to adopt the same crystal state converting liquid water into ice-nine and freezing it (real ice-IX has none of these properties, in the phase diagram above you can see that it only exists in a  narrow range of high pressure and low temperatures).

So, in the book there is a drama that develops to prevent ice-nine from "escaping" the lab and converting all liquid water on Earth to ice. The self catalyzing and spreading properties of ice-nine have been used as a metaphor for prions. Prions are "misfolded" proteins that, when they come into contact with the correctly folded form of the protein cause it to misfold as well. Prions are responsible for some forms of infectious diseases and can be difficult to inactivate when they contaminate a surface, and in some cases may even be passed through other living organisms like plants. Prions may also have other non-disease roles and might serve as a molecular "memory" for some fungi.

So, there are types of enzymes that cut specific DNA sequences resulting in a double-strand break. The cells has its own endogenous machinery to repair these breaks and often uses the sequence of the intact DNA strand as a template for repair (in diploids there are two copies of most DNA sequences and one can be used to repair the other). The trick is if the enzyme cuts the same position as it is inserted into the genome, then repair will copy the DNA sequence of the enzyme to the new DNA strand. In essence this converts a heterozygote (1 copy) into a homozygote (2 copies) and the gene is inherited by all of the individuals offspring rather than the normal 1/2.

drive
(The image above is from DiCarlo, J. E., Chavez, A., Dietz, S. L., Esvelt, K. M., & Church, G. M. (2015). Safeguarding CRISPR-Cas9 gene drives in yeast. Nature Biotechnology, 33(12), 1250–1255. doi:10.1038/nbt.3412)

This type of system has been demonstrated in a few organisms now. If it escaped the lab it could potentially convert the entire wild species world-wide to carry the genetic modification. In reality there would probably be some resistance due to genetic variation at the target site that is resistant to cutting by the enzyme and/or new mutations that result in resistance (double strand breaks can have a very high mutation rate when repaired, depending on the type of repair, see Non Homologous End Joining, NHEJ, and Microhomology Mediated End Joining, MMEJ).

This type of gene-drive system (there are diverse types of gene-drive and some do not have this property) might be though of as a genetic ice-nine. If it escapes the lab it could spread relentlessly and result in a permanent change to a wild species, which obviously raises a range of ethical issues. Actually I am surprised this analogy has not already been made (a Google search did not turn up any clear matches). The current hot technology to do this type of gene drive is known as CRISPR/Cas9, sometimes referred to as a Cas9 system, and there is an approach known as "in vitro CRISPR/Cas9-mediated editing" or "ICE" system---ICE-Cas9 is already almost, in words, ice-nine!

One journal article "Hammond, A., Galizi, R., Kyrou, K., Simoni, A., Siniscalchi, C., Katsanos, D., … Nolan, T. (2015). A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nature Biotechnology, 34(1), 1–8. doi:10.1038/nbt.3439" describes the use of this type of technology in a CRISPR/Cas9 approach to target and disrupt genes that lower female fertility. This publication caused quite a splash and so far I have been very quiet about it publicly. I was one of the peer reviewers of the article when it was submitted for publication; you can't discuss an article you are asked to review before it is published and generally the identity of reviewers is kept confidential---this mindset, I am realizing now, led to me avoiding going into details about the publication even after it was published. In this case there is the potential of using the technology to drive the mosquito Anopheles gambiae to, possibly, extinction by causing female infertility (the drive can still act in males and be inherited by all of a male's offspring including females which would then be infertile). In this scenario, at some point the last fertile female mates with a gene-drive male and there are no more fertile female offspring... In my review I urged caution, secure containment, and to not share the modified mosquitoes with other labs (contrary to the usual scientific custom). I also urged the authors to check on the possibility of resistant mutants arising by the repair of double stand breaks (which can have a high mutation rate and create resistant DNA sequences), and not to assume the population transformation would proceed without any new mutations. It is easy to urge caution at an early stage until the technology and implications can be further evaluated; however, diseases vectored by Anopheles gambiae are responsible for half a million human deaths a year---contributing the the ethical dilemma.

There may be no large negative results, from a general human perspective, if Anopheles gambiae were driven to extinction by genetic technology. Apart from the specific ethical questions of driving the extinction of and/or permanently genetically modifying this mosquito, the big worry in this case is unintended side effects and the unknowns associated with the technology and the broader ecological role of Anopheles gambiae. However, what about other species and other applications? At some point this type of technology might be used for economic convenience rather than unambiguous humanitarian efforts. And there is also the potential of intentional malicious use of the technology---something that I am quite concerned about when I go through various hypothetical scenarios as a mental exercise. While the precision (including species specificity*, which will become important in a moment) and flexibility of targets is new, the potential for threatening unintended consequences of genetic modifications in the wild is not. One  example is viral immunocontraception for invasive wildlife control. Rabbits have multiplied like crazy in Australia and have been disruptive to the environment as well as agriculture. People have investigated using viruses to infect the rabbits that are modified to contain part of a reproductive protein, ZP3, found on the surface of mammalian eggs. When the rabbits immune system fights off the viral infection it is also tricked into attacking the female reproductive system rendering the rabbits sterile. I have heard people that have worked in this field say that there was not thought to be a great deal of potential of unintended consequences if the virus infected Australian mammals because they are marsupials with quite different ZP3 DNA sequences, and the probability of a wild rabbit escaping Australia to another continent with the virus is exceedingly low. ...and this is the point where I pause in the story to see if people jump to the next conclusion... Humans live in Australia and have a ZP3 sequence that is more similar to rabbits; the probability of a human moving between continents is very high. To be clear, there is no evidence that genetically modified viruses have affected human fertility, and I know of no case where an immunocontraception application has jumped species boundaries; however, despite a low probability of occurring the possibility is serious enough to warrant careful consideration and unintended consequences in this case start to sound like another science fiction story, Children of Men.

  • In some cases this type of system is predicted to potentially spread across some species borders, but I don't want to go into the details of this here.

CRISPR's

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I think there are only two, maybe three, people left in the world that have not heard about the CRISPR-Cas9 system. It originates from a type of bacterial immune response but has been recruited as a genetic tool and has swept through the genetic engineering community in the last few years. In fact, one of the criticisms of our last NIH grant applications was that we were not using CRISPR's...  Here are some links to a few articles of wide interest regarding this technology.

DARPA, Gene Drive Technologies, and the ENMOD Treaty

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I just returned from a "gene drive" workshop at NCSU's Genetic Engineering and Society Center. There is a lot to talk about from the meeting. Here I want to focus on a couple of specific details that came up in reference to the military funding of genetic technology. The meeting was held under Chatham House Rule, so I cannot identify the people who made the original statements. Some of these were in group discussions and some of these were personal one-on-one conversations. As a brief, overly terse, background statement to describe a complex field: gene drive technology is a new emerging technology that is potentially very powerful and could be used for beneficial humanitarian and species conservation applications where other methods have fallen short in their long term effectiveness.

First of all, I was told that DARPA is interested in funding gene drive technology for environmental modifications. DARPA helps to develop new technologies for military applications.  This could be for both species conservation applications as well as preventing infectious disease (and also there is obviously the possibility of malicious hostile use in military applications but this was not brought up). Apparently a man named Dr. Jack Newman (link) is slated to become the program manager of mosquito gene drive technology at DARPA.

So---to be frank---I believe this is potentially a very bad idea for many reasons. The first is strategic. If these kinds of technologies are to ultimately be used for beneficial reasons they must be acceptable in some degree to the public so that they can become adopted and utilized. The Pacific Islands have a very negative track record of being used for testing grounds of new technologies. This ranges from classical bio-control releases of invasive species, to loss of traditional land to military activities, to, probably the most glaring example, nuclear testing in the Marshall Islands that displaced Native People and resulted in a region becoming uninhabitable from the resulting radiation (also note French nuclear testing, under protest, in Tureia, link). There is nested within the issues of the loss of self determination resulting from colonialism by many Western Countries across the Pacific. Like it or not, public perception is a very real force that cannot be ignored. The Three Mile Island accident in 1979 led to an effective moratorium on new nuclear reactor construction until 2012; however, many of these new projects have also been canceled with the more recent Fukushima disaster also playing a role. The public reaction to GM Crops has also had a very real effect on the laws surrounding the technology and adoption of the technology around the world including in the Pacific (e.g., the GM Taro and GM Papaya controversies in Hawai'i, link).* Right or wrong, in the Pacific, military funding of a new technology will be initially evaluated within the perspective of other military tests of new technologies and the effects this has had on the people of the Pacific Islands. Even more relevant to gene drive technologies, in the 1970's a World Health Organization project to test the release of sterile mosquitoes in India (to suppress the local population and limit the transmission of disease to humans) was shut down due to public perceptions that it might also be a secret military bio-warfare test (link, incidentally there are also some documents on WikiLeaks related to this).

In a broader ethical-moral sense (and this is very much a personal opinion from the perspective of a US citizen) are we comfortable with the military guiding and controlling the research that goes on in our country? This may sound like hyperbole; however, a comparison of the huge difference in the levels of US military funding (on the order of $610 billion) and National Science Foundation funding (on the order of $7 billion) is objectively dramatic. Advances in research depend on grant funding and support. Which technologies government funding agencies choose to support affects not only the advancement of these technologies but the direction they develop in and as a direct result the future applications of these technologies (the history of Project Orion is one example where limited funding sources and issues of potential military uses caused development focused on military applications yet ultimately stopped a line of scientifically promising yet controversial research, link).

Ideally, for gene drives technologies to be able to realize their potential in beneficial applications, they should be supported and developed by sources other than the military and private companies---and yes, this is strongly motivated by public perception as well as ethical principles. Scientific funding bodies as well as state and local funding have more of a long term potential benefit than is initially apparent. Furthermore, accepting funding from the military lends false support to continuing the objectively inflated funding of the military at the expense of government agencies devoted to scientific research (NSF and others); at the end of the day the military can say that it should continue to receive research funding because of the projects it has supported, but this comes with a social cost. Wouldn't it be better if NSF could make this statement instead without the social cost?

Okay, now comes the ace card that I have been hiding so far in this article... At the meeting, someone brought up (within the context of more "traditional" synthetic biology) that the US is a signatory to the ENMOD international treaty which came into force October 5, 1978. This treaty prohibits the military from using environmental modification technologies that have widespread and/or long-lasting effects. Interestingly, "Environmental Modification Technique includes any technique for changing – through the deliberate manipulation of natural processes – the dynamics, composition or structure of the earth, including its biota" (full text). This gets into philosophical discussions about the role of physical coercion by the military and the state, which I do not want to go into here (links for reference, military, monopoly on violence); however, I will appeal to the common-sense notion that military force by a state is a hostile act although this is more difficult to realize when it is done in a way that aligns with your own interests.  Since gene drive technologies are deliberate manipulations of natural biological processes with long term and possible widespread effects on manipulation of the environment, is DARPA military funding of gene drive technology even legal according to international treaty that the US agreed to support?

* I don't mean to sound overly pessimistic; there does seem to be a disconnect between the public-perception-of-public-perception and public-perception. When I talk to people about the type of work I am doing in the lab and the possibility of future applications at times I have been laughed at and told it will never be possible because people in Hawai'i are dead set against any genetic modifications. However, when I go into more detail about the non-native mosquitoes being modified to help protect native bird species and the self limiting nature of the genetic technology many of these same people are personally positive about the applications, but maintain that most people would be against it. Often the debate seems to be framed in terms of science versus people, especially native interests, with the recent debate over the telescope at Maunakea as an example (link), and it becomes all to easy to fall into overly polarized positions. GM Papaya and GM Taro (Kalo) are perhaps good examples of different ends of what is acceptable with genetic modification technology. GM Papaya is not without controversy but is now widely adopted and commercially important in Hawai'i; however, GM Taro was rejected in large part due to the importance of Kalo to traditional Hawaiian culture. As a diverse collection of cultures and values that does not always agree as individuals we can still have a public dialogue and make common choices; everyone has a role to play.