Important questions global health and science leaders should be asking in the wake of horsepox synthesis

The publication of the experimental work that synthesized horsepox is imminent, according to multiple reports. Horsepox no longer exists in nature, so this was the creation of an extinct virus in the same genus as smallpox. It doesn’t infect people, but causes pox disease in horses. Researchers have cited several objectives for the work, including the intention to develop it as a smallpox vaccine, the intention to develop it as a virus-based cancer treatment, and the intention to show that synthesizing smallpox de novo is possible.

The work raises a number of serious questions and concerns, partly about the specifics of the work and partly about what this says about biosecurity and biosafety considerations related to a circumscribed set of experiments.

The first question is whether experimental work should be performed for the purpose of demonstrating something potentially dangerous and destructive could be made using biology. In this case, horsepox was created in the laboratory, at least in part to show that synthesis de novo of smallpox virus is feasible. In this specific case, leading virologists have agreed for many years that de novo smallpox synthesis was scientifically feasible, and there has been no serious counterargument that it was not feasible. But the important decision going forward is whether research with high biosafety or biosecurity risks should be pursued with a justification of demonstrating that something dangerous is now possible. I don’t think it should. Creating new risks to show that these risks are real is the wrong path. At the very least, it’s a serious issue needing serious discussion.  

A second question that is more relevant to this experiment is how much new detail will be provided in the forthcoming publication regarding how to construct an orthopox virus. It is one thing to create the virus; it’s another thing altogether to publish prescriptive information that would substantially lower the bar for creating smallpox by others. The University of Alberta lab where the horsepox construction took place is one of the leading orthopox laboratories in the world. They were technically able to navigate challenges and inherent safety risks during synthesis. Will labs that were not previously capable of this technical challenge find it easier to make smallpox after the experiment methodology is published? 

A third question relates to the approval process for experimental work with implications for international biosecurity or international biosafety. The researchers who did this work are reported to have gone through all appropriate national regulatory authorities. Researchers who created horsepox have said that the regulatory authorities “may not have fully appreciated the significance of, or potential need for, regulation or approval of” this work. So while work like this has potential international implications – it would be a bad development for all global citizens if smallpox synthesis becomes easier because of what is learned in this publication – the work is reviewed by national regulatory authorities without international norms or guidelines that direct them. This means that work considered very high risk and therefore rejected by one country may be approved by others. 

In the case of the horsepox experiment in Canada, the Advisory Committee on Variola Virus Research at WHO was briefed on the work after it was completed. Moreover, the primary charge of that committee is actual smallpox research itself (as opposed to horsepox). Beyond that, this WHO committee is unique. WHO does not have special disease by disease committees that review work on a case by case basis for other pathogens.

I think the new P3CO policy published by the White House in January 2017 could be a good step forward in the US regarding future policy development for experiments involving potential pandemic pathogens. Whether and how that policy will be implemented remains to be seen since it is guidance for federal agencies but does not require their action. Importantly in this case, even if this policy had been implemented in the US, it doesn’t seem that the policy would have had bearing on the horsepox research had that been proposed in the US. So even as the US has spent a substantial amount of time considering these kinds of issues, it still doesn’t have policy (or high-level review committees) that directly considers experiments like this. Beyond that, there is no international component to P3CO. There clearly needs to be an international component to these policies. We need agreed upon norms that will help guide countries and their scientists regarding work that falls into this category, and high-level dialogue regarding the necessary role of scientific review, guidance, and regulation for work that falls into special categories of highest concern. It is not clear that these considerations are now even being discussed internationally.   

The rapid advance of biology in the world overall will continue to have enormous health and economic benefits for society. The entrepreneurial and unpredictable nature of biological research, now coupled with powerful global markets, is overwhelmingly positive for the world. But this case of horsepox synthesis shows us that there are also specific and serious challenges that require special attention now.  

Tom Inglesby, MD, is the director of the Johns Hopkins Center for Health Security

The Rewriting Revolution

Earlier this week, I discussed the ethical and safety implications of the potential for germline editing using CRISPR/Cas9 technology, referencing  the ‘rumors’ that international scientists had already begun experimenting with human embryos. On Wednesday, the rumors became fact, as Nature News reported the world’s first example of gene editing in human zygotes. The study, led by Junjiu Huang at Sun Yat-sen University, was published in the online journal Protein and Cell.

The scientists targeted the human beta globin gene (HBB) which, when mutated causes the blood disorder b-thalassemia. The investigators used non-viable embryos; eggs that are fertilized by two sperm and therefore do not develop. They are typically discarded by fertility clinics, providing a potential ethical alternative model system for normal human embryos.  While CRISPR/Cas9 was shown to be successful in cleaving the endogenous HBB gene, the paper details numerous obstacles to using this technique for clinical applications in human embryos.

First, the efficiency of the technology was found to be much lower than what would be necessary for use in normal embryos. The researchers injected 86 embryos with Cas9, guide RNA to target HBB, and single-stranded DNA (ssDNA) that, if successful, would be inserted into the HBB locus. Seventy-one of these embryos survived. Out of these, 28 were cleaved by Cas9, but only 4 or 14.3% were edited with the ssDNA. Instead, the double-stranded breaks induced by Cas9 were preferentially repaired by direct ligation of the broken ends, which would not correct the mutations and could also induce additional mutations into the target gene. For ethical reasons, the efficiency and survival would need to be close to 100% to be used in viable human embryos.

Second, numerous off-target effects were detected, meaning the guide RNA induced double-stranded breaks in other genes besides HBB. These unintended mutations were seen at much higher rate than what had been observed in stem cells or mouse embryos, which is alarming. Off-target editing can damage healthy DNA to cause numerous side-effects and diseases if propagated in human embryos.

The results of this study further underscore the need to halt the use of CRISPR/Cas9 in human embryos until the technique can be optimized in human cells or animal models to improve the efficiency of gene-editing and reduce off-target mutations. However, rumors continue to suggest that at least four other groups are editing human embryos. 

Designer Babies “R” Us

Asilomar and Napa, separated by 150 miles, are now linked as meeting sites for scientists to discuss the self-regulation of biotechnology. In Asilomar in 1975, the topic was recombinant DNA technology, or the joining of DNAs from different organisms. After discussions on the safety and risks, scientists and physicians voluntarily produced governing principles for their work, including a list of prohibited high-risk experiments. In January 2015, scientists again gathered in California, this time to discuss CRISPR-Cas9 technology, concluding that there is an urgent need for continued open discussion on its use for human genome modification.

The CRISPR-Cas9 system is utilized by bacteria to protect themselves from infection by viruses. Upon detection of invading DNA, the bacterium produces two types of short RNA, one of which matches the viral sequence. This guide RNA forms a complex with Cas9, a nuclease that can cut DNA upon binding. The complex can then bind to the complementary virus DNA target site, leading to its excision and degradation, thereby disabling the virus. This bacterial system has been co-opted by scientists for use in genomic engineering to change exogenous DNA in a variety of cells and animals. Mutations can be introduced by targeting genes-of-interest for excision by the complex, simplifying the creation of gene-knockout models to examine gene function. However, the technique can also be used to repair mutations by adding DNA segments carrying the correct genetic sequence along with the CRISPR-Cas9 complex. After excision, the cell will use this DNA as a template for its repair machinery, thereby introducing the ‘healthy’ sequence into the genome. If this is done in reproductive cells, the CRISPR-Cas9 system has the ability to modify human embryos, an application that has enormous ethical implications.

By editing the DNA of the egg and sperm cells, called “germline engineering”, it could be possible to correct mutations that cause diseases. For example, a researcher described the potential to harvest the eggs from women carrying the BRCA1 mutation, a dominant mutation that predisposes women to breast and ovarian cancer. CRISPR-Cas9 would be used in these eggs to correct the mutated DNA and create a viable embryo with the ‘healthy’ BRCA1 gene. This genetic change would then be passed on through future generations. However, the technology is still in its infancy – scientists do not yet know the potential for off-target effects, or even on-target editing with possible side effects. Furthermore, the width of its potential application is troubling to bioethicists. With recent advances that further streamline the system, the possibility of designer babies that could alter the scope of human evolution is more reality than science fiction. In fact, rumors abound that scientists outside of the United States have already submitted papers for publication that detail their use of CRISPR in human embryos.

Given the lack of regulations on germline engineering in the United States, Jennifer Doudna, the molecular biologist who helped develop CRISPR, convened scientists, ethicists, and law experts to discuss the concerns. The group detailed a number of steps to be taken, including: discouraging attempts at genome modification, creating forums for continued discussion, encouraging transparency of research to evaluate the safety of the technology, and finally the convening of a global group to openly discuss the merits and risks and recommend policies.

The meeting at Napa was essential to begin the conversation on CRISPR-Cas9 technology and attract public attention to these issues. However, there are many additional measures that need to be addressed in further discussions. Notably, and in contrast to the original Asilomar, the researchers did not call for a moratorium on the controversial germline engineering, instead only “strongly discouraging” the work. Until the safety and efficacy of CRISPR in germline editing is determined, a self-imposed pause on altering human eggs, sperm, or embryos, should be in place. It is possible the recent government-imposed ban on funding for the gain-of-function (GOF) studies on influenza and SARS viruses led to the scientist’s reticence to impart additional restrictions on their community. In contrast though, GOF studies have broad biosecurity implications - the escape of a virulent strain has the potential to affect the general public, while germline engineering affects only the individual and his or her progeny. Therefore, it remains to be seen what regulatory approach the U.S. government will elect to take regarding genome editing using the CRISPR-Cas9 system.

However, this raises an additional point that was not defined in the Napa meeting: Who is responsible for leading the discussions on the technology moving forward? Should it be left exclusively to the research community, or done in conjunction with national and international regulatory bodies? An overhaul of the current laws and legal system may be necessary in light of the uncertainty of the role that the U.S. government should play in regulating biotechnology. A potential model is the United Kingdom and their response to the issue of mitochondrial DNA replacement therapy, which is another form of germline modification. The procedure was banned while the government sponsored numerous scientific and ethical studies. After these demonstrated the safety of the technique, the House of Commons and Lords voted to approve the technique, albeit under strict regulation. A similar protocol may be useful for the U.S. and abroad to evaluate CRISPR-Cas9, in order to ensure the technology is used safely for germline editing.