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Discussing gain-of-function research

posted Sep 8, 2015, 1:06 PM by Kevin Esvelt   [ updated Oct 25, 2016, 9:28 AM ]
In the course of promoting safeguards and transparency in the field of gene drives, many have drawn a comparison to so-called “gain-of-function” (GOF) research on influenza and other potentially pandemic pathogens. Like gene drives, GOF has the potential to affect people outside the laboratory if something goes wrong. I haven't previously touched on the subject, but Marc Lipsitch and Thomas Inglesby invited me to join them in highlighting the need to engage those most likely to be affected – in this case the clinical community, which has heretofore been largely silent on the issue – and holding up CRISPR and gene drives as an example to follow. Our work was published today in the Annals of Internal Medicine.

As an evolutionary biologist, my skepticism of GOF influenza research should not be surprising. These studies typically seek to evolve more virulent strains of influenza in the laboratory in order to provide advance warning of which mutations we should look out for in the wild. They are controversial because they deliberately create the very agents we fear, and by this point we know that mistakes are inevitable when relying on barrier confinement. Perhaps not this year, or this decade, but they will occur.

Yet there is also a cost of doing nothing. The question is whether the risk is worth it. Is the knowledge of which mutations we should fear sufficiently valuable?

First, it is not at all clear that advance warning that a virus is near to becoming a pandemic would allow us to do something about it. Second, we have abundant evidence that evolution is stochastic. That is, if we run an evolution experiment in the laboratory many times under conditions as identical as possible, the iterations will frequently come up with different solutions. This is true whether one is evolving individual proteins or entire genomes. The resulting behavior of the evolved variants may be functionally the same – especially for whole-genome evolution studies where many genes are evolving in concert – but the underlying mutations producing that outcome commonly differ. This certainly came up in our PACE study examining evolutionary pathways and stochasticity. It's not true for every system, but does seem to be the case for many. Precisely why this would be different for influenza is not clear.

I am consequently not at all concerned by the current moratorium on funding gain-of-function studies. In my view, the odds of discovering which mutations to fear – and being able to use that knowledge to prevent a pandemic – are very slim, and consequently not worth the attendant risk of accidentally causing the same pandemic we seek to prevent.

Not only might a human-created accidental pandemic lead to many unnecessary deaths and suffering, we must also consider the effects of such an event on public confidence in scientific research more generally. In a post-pandemic world, it would be very easy to question why the government should fund life scientists at all if their judgment was so poor as to knowingly risk creating the pandemic that killed a million people. This goes double when superior safeguards were available but were not employed. In short, reprise all the public-trust reasons why we should take stringent precautions to avoid an accidental gene drive release, then add the far more harmful consequences of a pandemic human virus.

Yet there are other reasons to perform gain-of-function studies. The Kawaoka laboratory just published research identifying superior influenza viral backbones for rapid vaccine production. This is important because current influenza vaccines require us to generate of large quantities of virus, which is subsequently inactivated and injected to provoke an immune response to the viral proteins.

Historically, we generated those quantities by leveraging chicken eggs – which we have in abundance – as growth chambers. Yet egg-based vaccine production is slow, vulnerable to disruptions in the egg supply, can select for mutations specific to replication in eggs, and typically produces viruses that are contaminated with egg proteins capable of causing allergic reactions in sensitive people. There is consequently a movement to switch influenza vaccine production to cultured mammalian cells.

The problem is that vaccine virus yields are quite low in cultured cells. For some vaccine candidates, yields can even be low in eggs. The researchers consequently sought to evolve a backbone capable of efficient virus replication in cultured cells. They succeeded.

On a technical level, this was a truly impressive work. Using library generation and screening, generating known mutations from the literature, and combining these hits to maximize viral replication required a tremendous amount of dedicated effort. The resulting backbones increased production in both mammalian cells and also in eggs, in some cases by up to 200-fold. As is usual for directed evolution experiments, the mechanism remains unclear.

This work was performed before the US government imposed a moratorium on gain-of-function research until an independent assessment could evaluate safety. But the new backbones did marginally increase the severity of disease in mouse models, if only marginally, so the experiments producing them qualify as GOF. Predictably, the study is being held up as an example of why the moratorium was a mistake or at least applied too broadly - though the director of NIAID maintains that an exemption would have been granted.

Why specifically an exemption would have been granted in this case is not clear, but it may have had to do with the fact that the researchers used a form of intrinsic confinement. Specifically, they altered a sequence in the hemagglutinin gene involved so that the resulting viruses were of low pathogenicity and consequently exempt from Select Agent status. They did not specify how many known mutations were required to regain pathogenicity (obviously there may be unknown mutational routes as well). It's certainly better than no intrinsic confinement.

But it may not be as good as alternative confinement approaches that could make all influenza virus research safer, and is certainly not as good as stacked confinement strategies. What might those be? While I'm not a practicing virologist, several do come to mind. For example, researchers could insert a sequence into the viral backbone that would be targeted by RNAi in human cells, then produce the virus in non-human cells. Since the goal is to generate inactivated virus, you don't need it to replicate in human cells to generate the immune response. Alternatively, produce it in human cells with the relevant RNA knocked out in order to permit viral replication specifically in those cells. There's this technology called CRISPR that makes it easy to do such things now...

If the RNAi trick doesn't work for some reason - this is biology, after all - there are other options. One might instead encode one of the viral proteins in the genome of the producing cell and delete it from the viruses being tested, thereby ensuring that the virus can only replicate in cells containing the missing component. Sure, it's possible that the viral replication cycle might require protein production to be delicately timed and responsive to the viral copy number. If so, this could be corrected using a trans-splicing intein to assemble one of the viral proteins once each half is produced. Yes, the devil is certainly in the details, but the bottom line is that there are several potential ways of incorporating intrinsic confinement.

While these aren't necessarily trivial endeavours, the authors of this work are clearly highly skilled; I am confident they could succeed. Once available, the safeguards could then be used for other types of experiments with these viruses, dramatically reducing the chance of accidentally causing a pandemic. The resulting risk reduction might be sufficient to make these kinds of studies worth pursuing. I personally would be much happier if someone tried.

One last point. According to Science News, “the researchers contend that their findings may help bring future pandemics under control faster.” This is indeed a critical challenge – I would prioritize the ability to quickly make and scale up a vaccine to any given virus extremely highly – but the approach still assumes that we will be able to make a suitable vaccine strain in the first place. That itself can take time – at least, if one relies on the current paradigm. This is an excellent reason to move beyond the current paradigm.

Instead of simply injecting killed virus into patients and waiting for the appropriate immune response, we might use vectored immunoprophylaxis (non-integrating gene therapy) to instruct cells to produce the appropriate antibodies. This approach has the greatest potential for a quick turnaround, as it only requires identifying functional antibodies from the first infected individuals, screening them for binding to the virus (or other agent) to isolate the most protective ones, then making lots of DNA encoding those antibodies for delivery into patients. In principle, you could start cranking out “vaccine” inside of a week and quickly scale up much faster than current vaccine production allows.

The overall lesson is that we should look further ahead towards more broadly applicable technologies. Investing in better safeguards can reduce risks across the board, potentially enabling highly beneficial experiments that would otherwise be too risky. And developing new technologies can offer benefits – in this case, truly rapid response to new and dangerous viruses – that current approaches could never hope to match. Combined, these two advances could enable swifter (albeit still highly flawed) detection of potentially dangerous viruses and let us do something about it.

Tags: engagement, transparency, responsibility, innovation, gain of function