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Researchers Slam 'Stupendously Unethical' CRISPR Germline Genome Editing Experiments

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NEW YORK (GenomeWeb) – Southern University of Science and Technology's He Jiankui ignited a firestorm of epic proportions at the Second International Human Genome Editing Summit in Hong Kong this week when news broke through the Associated Press and through a series of self-produced YouTube videos that he had used CRISPR-Cas9 technology to disable the CCR5 gene in the germline genome of twin embryos in order to make them immune to HIV.

The father of the twin girls, nicknamed Lulu and Nana, was HIV-positive and the mother was HIV-negative, He revealed. The embryos had been successfully implanted, and the babies had been born seemingly healthy.

Widespread condemnation

The news — which initially was not accompanied by any kind of data or published research — almost immediately drew near-universal condemnation from the scientific research community. Though He has been on unpaid leave from the Southern University of Science and Technology in Shenzen, the institution said the work violated its codes of conduct and called for an investigation. China's National Health Commission has also ordered officials to investigate He's claims, and Shenzen's Health and Family Planning commission said it plans to look into He's work.

Direct human experimentation [with CRISPR] can only be described as crazy.

More than a hundred Chinese researchers wrote an open letter criticizing the work, saying that "direct human experimentation [with CRISPR] can only be described as crazy," and calling He's experiment a "huge blow to the global reputation and development" of Chinese research.

In the US, meanwhile, Rice University said it has launched an investigation of one of its researchers, Michael Deem, who told the AP he helped He with his project. Deem was He's advisor when He was in graduate school. In a statement, Rice said it had no knowledge of the work, that none of the research was conducted in the US, and that "this work as described in press reports violates scientific conduct guidelines and is inconsistent with ethical norms of the scientific community and Rice University."

US National Institutes of Health Director Francis Collins released a strongly condemnatory statement on Wednesday, saying that the experiment "represents a deeply disturbing willingness by Dr. He and his team to flaunt international ethical norms." Collins rejected every aspect of the work, from its secrecy to what he called He's "utterly unconvincing" justification of medical necessity for deleting the CCR5 gene in the embryos.

"It is profoundly unfortunate that the first apparent application of this powerful technique to the human germline has been carried out so irresponsibly. The need for development of binding international consensus on setting limits for this kind of research, now being debated in Hong Kong, has never been more apparent," Collins added. "Without such limits, the world will face the serious risk of a deluge of similarly ill-considered and unethical projects. Should such epic scientific misadventures proceed, a technology with enormous promise for prevention and treatment of disease will be overshadowed by justifiable public outrage, fear, and disgust."

The American Society of Human Genetics also reaffirmed its 2017 position statement on human germline genome editing, supporting publicly funded in vitro research into the potential clinical applications while outlining the necessary scientific and societal steps that need to be taken before such clinical applications can take place.

"It is premature to perform germline genome editing that culminates in human pregnancy," ASHG President David Nelson said in a statement this week. "Important scientific, ethical, and policy discussions are taking place, in a variety of venues within and outside ASHG, but many vital questions remain unanswered."

Most importantly, the voices of CRISPR experts are ringing out, loud and clear. The University of Berkeley's Jennifer Doudna issued a statement calling on He and his colleagues to explain why they chose to "break from the global consensus that application of CRISPR-Cas9 for human germline editing should not proceed at the present time," adding, "It is essential that this news not detract from the many important clinical efforts to use CRISPR technology to treat and cure disease in adults and in children."

The Broad Institute's Feng Zhang called for a moratorium on the implantation of edited embryos, at least until the scientific community has had a chance to come a consensus about safety requirements. Like Collins, he also called out He's lack of transparency.

In his own statement, the Broad's David Liu called the work "a serious breach of ethics that I hope will serve as a wake-up call for the community." Though embryonic editing may be justifiable at some point in the future after careful consideration of the potential benefits, risks, and ethical issues, Liu added, this experiment doesn't meet these standards and shouldn't have been conducted.

So far, the only prominent researcher to come to He's defense has been Harvard's George Church, who told the AP that the investigator's goal was "justifiable," given the public health threat posed by HIV. 

He's explanation

For his part, He seems to feel his actions were right. In a presentation at the summit on Wednesday, the researcher apologized for the unexpected nature of the news, but admitted that he had been working with the AP for months to report on the study and said his university had not known about the work.

He outlined the impetus for his work, noting that a natural mutation known as the CCR5Δ32 deletion — which is carried by about 10 percent of the population in certain countries in Europe — confers profound resistance to HIV1, and walked the assembled researchers through his early experiments doing CCR5 knockouts in mice, designing guide RNAs for CCR5 in human cell lines, and then conducting experiments in monkeys.

"We found that injecting Cas9 closer to fertilization promoted the most efficient editing efficiency, consistent [with other findings]. This also reduced mosaicism," He said. "To look more closely on the mosaicism, we also sequenced every individual cell in several embryos. Editing appeared to occur at the 1-, 2-, and 3-cell stage. Under the assumption that Cas9 degrades quickly and requires time to find the right target, we explored a strategy to reduce mosaicism by delivering a second injection of Cas9 to an embryo at the 2-cell stage."

At that point, He said, his team looked to see if the protocol could be transferred to human embryos. They assessed whether the editing had created any off-target effects by performing single cell whole-genome sequencing on the embryos, prior to implantation. They also used a specially designed program to analyze the parents' genomes in order to detect possible "novel risk sites unique to each embryo, which emerge from inherited SNPs," He said. "We were able to visualize the personalized off-target hotspot pool, on the order of 10,000 sites per embryo. We used whole-genome sequencing to assess for these spots and validated any findings by Sanger sequencing."

When they looked for off-target effects in the human embryonic stem cells, He said, the researchers detected one off-target effect in an intergenic region, 279 kilobases away from any known genes and not near any known non-coding RNA or transcription factor binding sites. They couldn't tell whether this was an inherited mutation or caused by editing. In one embryo, the team identified a 6-kilobase deletion at the target site, which did not affect any gene other than CCR5.  

"Pre-implantation genetic diagnosis showed two sites were edited. One was a frameshift lockout, which should shorten the CCR5 protein similar to the natural protective variation," He noted. "Another had a deletion, which was expected to maybe destabilize local protein structure around the nearby HIV1 binding site."

After birth, deep sequencing of the cord blood confirmed the editing pattern the team saw in PGD testing, but neither deep sequencing nor Sanger sequencing detected the intergenic off-target effect that was observed during PGD testing, He said, adding, "This suggests that this was an artifact of a single cell amplification, or a mosaic off-target that happened to occur in the few blastocyst cells assembled for PGD."

He also noted that the parents were informed of the implications and chose to implant the embryos regardless of the risks, and that the researchers plan to continue their assessments of the twins, including blood tests for HIV infection potential and further investigations of off-target effects and mosaicism in multiple tissues, for the next 18 years. He also hoped they would continue to accept support as adults.

But despite what may sound like a typical presentation at a scientific conference, this was anything but normal.

Among the many details about his work, He had also noted that eight couples were enrolled in his trial — one eventually dropped out, but seven were still involved. All the fathers were HIV-positive. Further, he said that about 30 total embryos had been created and that 70 percent were edited.

And although the trial has been put on hold, He said, he revealed that there may be an additional pregnancy. It's still too early to tell if it's viable.

For this specific case, I feel proud.

The detail that seemed to cause the most uproar was He's own attitude toward the work — "For this specific case, I feel proud. I feel proudest because they had lost hope for life," he said about the parents of the infant twins. He called CCR5 editing an "unmet medical need," citing the lack of an HIV vaccine, and said this work could potentially have a positive impact on the lives of "millions of children."

Unknowable complications

But HIV researchers seem to disagree. In the case of the twin girls Lulu and Nana, only the father was HIV-positive and, according to He, his viral load was undetectable. Further, the researchers washed the father's sperm in order to prevent transmission of the virus.

These circumstances alone would have prevented the virus from being passed to the embryos, obviating the need for any kind of gene editing, Martin Markowitz, clinical director of the Aaron Diamond AIDS Research Center at Rockefeller University, told GenomeWeb.

I think that what he did is eugenics research. This whole thing stinks.

"So, the idea is that he was protecting these babies from HIV infection from their father? Well we know very clearly now that the chances of this HIV-infected father infecting the mother is probably zero if he's got a viral load that's undetectable for six months," Markowitz said. "So, I don't even understand what this is all about. I'm a little bit confused as to how this is going to lead us to an HIV vaccine. He called it HIV vaccine research, but I don't think that's the case. I think that what he did is eugenics research. This whole thing stinks."

Indeed, Markowitz added, instead of helping Lulu and Nana, He might have caused another problem for them. "If he has generated twin girls who are CCR5-deleted, the question is what will happen to them as they age," he said. "Because I don't think anybody has any clue at all as to whether he has hurt them. And it's indeed possible that he has made them more susceptible to other infections such as West Nile and other viral illnesses."

He is certainly not the first researcher to hypothesize that deleting CCR5 with CRISPR could benefit HIV patients. A cursory PubMed search turns up dozens of recent studies along those lines: a March study from a team in Brazil in Genetics and Molecular Biology compared the effects of CRISPR-Cas9 editing to TALENs on the CCR5 gene; a July paper from researchers in China in Reviews in Medical Virology discussed the development of CRISPR as a tool for HIV1 treatment; and a paper published in Molecular Therapy Nucleic Acids in September described the off-target effects of inducing CCR5Δ32 homozygotes through CRISPR editing in a human cell line.

These studies all use words like "in the future" and "has promise" to describe these approaches. The researchers are working with cell lines and animal models. In some cases, they're trying to determine which approaches have the best editing efficiencies, and certainly there are still many questions remaining about the safety of germline genome editing. This is what seems to irk researchers most about He has done.

"In the sense of what we know about HIV, [CCR5 as a target] makes sense. But the context is all wrong," said Ronald Swanstrom, a principal investigator and virology researcher in the biochemistry and biophysics department at the University of North Carolina at Chapel Hill. "You just wouldn't want to do this on a person-to-be under these circumstances. The potential benefit is so small, and the risks are so unknown, that it just doesn't add up as making any kind of sense at all."

Like Markowitz, Swanstrom also cited the increased risk of susceptibility to West Nile virus and other RNA viruses for the twin girls. "That's now a permanent feature of that person," he added. "It's perhaps a small risk, but their risk of getting HIV was also pretty small so it doesn't seem like a good trade-off."

Further, Markowitz noted, although there is one drug on the market that inhibits CCR5 — maraviroc, sold by Pfizer as Selzentry — it's not in widespread use because "it's not a particularly useful drug given the other treatments that are available."

For Swanstrom, however, the bigger risk is the unknown hazard posed by the off-target effects. Researchers have observed the effects of off-target mutations in smaller animal models, he said. In humans, who live longer and have bigger, more complex genomes, the risks are as-yet unknown.

And if the children end up with health problems, the question of who is responsible for their healthcare into adulthood is weighing heavily on the minds of researchers and ethicists. "Let's say it didn't work and the child was born with illness, or birth defect, or some congenital problem," Arthur Caplan, a bioethics professor at NYU School of Medicine, said. "Who's responsible for covering the healthcare costs? Him? You want to settle those issues before you start marching off to germline genetic engineering. Who's liable here?"

Ethical quandary

It's clear that there are many sources of anger over He's experiment — the work itself and the danger it poses to the children who have now been born is a very large part of it, certainly, but it is only one part.

There is also a danger to science itself. Whether researchers are fascinated by the sheer mechanics of the experiment, or whether they feel that germline editing should be a goal to work toward or be avoided altogether, they seem to universally agree that what He did was entirely unethical.

"It's fair to say He's not some kind of crackpot or kook. I think he did one of the most important experiments in the history of human genetics. Germline engineering to change a trait in children — that's a major, major experiment," Caplan said. "But it's stupendously unethical."

Caplan believes that human germline genome editing should eventually be allowed. At the genome engineering meeting in Cold Spring Harbor in August, he took part in a panel discussion on the topic in which he argued against blanket prohibitions on the method.

It's stupendously unethical.

"But you want to make sure you've got more animal work, you want to have more lab studies. [He's work] was way premature in terms of trying it in a human embryo to give birth to a baby," he said. "I think it does makes sense to try it on somatic cells and see how that goes. That's what makes it very unethical when you're doing something to children who can't consent. You've got to really try to minimize the risks. This experiment didn't do that."

In fact, Caplan concurred with Markowitz's view that He's experiment was "eugenic" in nature, especially as there are already ways to protect embryos against the transmission of HIV and treatments for the disease already available.

Further, there is great uncertainty over whether the subjects involved in the experiment truly gave their informed consent or understood the consequences of the experiment. In his presentation, He said the couple was highly educated, that his team spent more than two hours with them initially, and that he then spent more than an hour with them going over the informed consent form paragraph by paragraph. In answering a question from the audience on whether he or his team had received training on taking consent from trial participants, He noted that he consulted guidelines from the US National Institutes of Health.

But that doesn't seem satisfactory to many researchers. "Whether or not he had what we in the US would consider adequate oversight by an institutional review board is not clear," Markowitz said. "Whether the people who actually volunteered to do this, whether these people actually did even volunteer for this, whether it was done without them having a clear understanding of what was being done [is also unclear]." 

Indeed, the AP article noted that the consent forms called the project an "AIDS vaccine development" program.

UNC's Swanstrom agreed, adding that because the source of He's funding is unclear, it's also unclear who oversaw the use of human subjects in this experiment. "If someone's going to do stuff like this, there ought to be a broader understanding that it's going on, if it's going to be done in the name of research and in the name of science," he said. "Then there's a lot of infrastructure that we have that comes into play when you start doing human subjects research. And one gets a sense, without knowing the details, that these things broke down."

The sum of all the uncertainties, the lack of transparency, and the intentional secrecy have also greatly contributed to the skepticism towards He. "This is not really the way we do things in science," Markowitz said. "If we have things that are earth-shattering, they generally appear in peer-reviewed publications, and something like this certainly would appear in a journal like Nature, or Science, or something equivalent. So, to have presented this the way it has been makes one sort of step out and go, 'Really?'"

NYU's Caplan said he was particularly annoyed by something He said to the AP: "I feel a strong responsibility that it's not just to make a first, but also make it an example. Society will decide what to do next."

That's backwards, Caplan said — first, society has a discussion and decides what to do about something and then researchers do the experiments. "You don't do it and say, 'Haha, figure it out.' Cloning was done that way — kind of quiet and then here's Dolly the sheep. It can uncork a tremendous amount of blowback that sets back the whole field. People say, 'Well, these renegade guys, we don't know what the hell they want, we better make sure there's no money for it or we better discourage it.' And the experience with cloning is instructive about why you don't just say, 'Hey, I'm going to do something,' and that's it," he added. 

Indeed, there are now fears that funders may decide to step back from gene editing science as a whole, even as the field is gaining huge momentum.

"He has taken technology and misapplied it in a very questionable way and I think has violated some very basic principles of human research, of applying science responsibly, and even applying science in a way that benefits mankind," Markowitz said. "And I hope that he doesn't set the field back instead of advancing it, because somatic editing may indeed be important for people or families who do have genetic diseases that perhaps may be amenable to such types of editing. And when I see stuff like this, it makes me upset, and I just can't say it any stronger. I doubt that you will talk to a scientist or an academic physician that would feel differently."

As for He's data, he noted after his presentation this week that his paper is currently being edited and that he plans to send it to additional experts for further comment, though he has declined so far to submit it to bioRxiv.

But as far as Caplan's concerned, no reputable scientific peer-reviewed journal should publish the paper. "You want to condemn this guy. I think he should put something online [but] I think journal editors should make it clear that if you don't follow appropriate regulatory requirements, then you don't get published," he said. "Let the editors speak up and say [they] wouldn't publish, today or in the future, his paper or anything like it if it's out of conformity with agreed-upon standards in informed consent, review by committee, consistent with national policy, that sort of thing."

Caplan also called for a meeting or conference of experts to agree upon and issue guidance for enforceable international standards on germline genome editing — currently, there aren't any, he noted.

"In the history of human experimentation — even if you're first and even if you kind of achieve a goal — if you're way out of step with the morality of the community, you don't get to be a hero," Caplan concluded. "You wind up being sort of this odd duck."

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