√ Who Leaked Mitalipov Crispr Human Embryo Paper?

The new CRISPR human embryo paper from Shoukhrat Mitalipov is stirring things up, but then there’s also the murky back story as to how the news of this as yet unpublished paper got leaked in the first place. The actual paper is still not out and from what I understand hasn’t even been officially released by Nature in embargoed form to journalists. So what the heck happened?

Specific details of the Mitalipov paper popped up a couple of days ago on Tech Review and on a UK news outlet (both by the same author, Steve Connor) perhaps as much as a full week before the paper is set to come out. Leaks at the White House and leaks in science?

You can see the “iNews” front page at right. Connor’s piece in Tech Review in my view was a bit too upbeat about the manuscript in terms of “safety” in particular, but I haven’t even seen the manuscript so I can’t be sure and Connor’s scoop on this admittedly was interesting. I’m excited to read the actual CRISPR human embryo paper too and without it much of what is out there remains somewhat unclear.

We all want to know more about the data, but many seem now to be asking the same kind of bigger picture question too, ‘who leaked it?’

In his iNews piece, Connor quotes at least one anonymous source:

“Although Mitalipov and his colleagues are under a strict confidentiality agreement with a leading scientific journal, which has scheduled to publish the work next month [August], we understand from other sources that the study breaks new ground in demonstrating the feasibility of creating genetically modified babies. “I’ve heard Mitalipov has done it. He’s successfully done genetic modification of human embryos. The quality of the work was high,” said one senior scientist who wished to remain anonymous.”

Connors also quoted a Salk Institute scientist, apparently a co-author with Mitalipov, by name about the paper in the Tech Review piece:

“Reached by Skype, Mitalipov declined to comment on the results, which he said are pending publication. But other scientists confirmed the editing of embryos using CRISPR. “So far as I know this will be the first study reported in the U.S.,” says Jun Wu, a collaborator at the Salk Institute, in La Jolla, California, who played a role in the project.”

I asked myself, “How would anyone know to contact this one particular person Jun Wu regarding an unpublished paper not even released by the journal in the first place?”

It’s also notable that Connor’s quote says “scientists” as in plural.

The apparent fact that Nature has not even officially released embargoed copies of the paper to the press and won’t do so until early next week  means that no embargo was broken since no embargo yet exists (weird situation, huh?), but something unusual happened here.

I reached out to Connor, but he was unable to discuss sources regarding this story, which is totally understandable.

Does it really even matter if an important science paper or its key findings are leaked out a few days or a week in advance? If it does matter (and gut feeling is that it does on some levels), what are the risks to science and scientists? Or is it more about the journal itself maintaining control of the timing and the initial media coverage?

What do you think?

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√ Review Of Mitalipov Paper Crispr’Ing Human Embryos: Transformative Work On The Edge

In the same way perhaps that some excited relatives or parents-to-be both gush and worry about a baby before it is even born, our field has been transfixed for a week by the Mitalipov paper on CRISPR’ing human embryos even though the paper just now came out.

Figure 3a Ma, et al. Nature, 2017

Now that the paper is out, we can take a closer look at this “baby” and for us scientists that involves giving it a critical review. In science, “critical” often means a thorough once over with a somewhat skeptical eye, but not necessarily a negative one. Indeed, my overall take on this paper is positive. It is quite strong technically and has many elements that are innovative even though 3 previous studies have already tried CRISPR gene editing in human embryos of various kinds.

This new paper “Correction of a pathogenic gene mutation in human embryos” is in a different category than the other ones in its approach and implications. It is quite rigorous and contains generally very thorough analyses. There are still some very important open questions and I believe there are some issues with perhaps some small overstatements, but by and large this paper is top-notch.

What are the key take homes from this study? Let’s look at my big five questions and now some attempts at answers.

1. Off-target activity? They didn’t detect any. Overall some statements in the paper are perhaps a bit overexuberant such as statements of “abolished mosaicism” (they actually did find a mosaic embryo). I also do not believe they can be quite so confident about “no off-target activity”, when as best as I can tell they did not look thoroughly in enough embryos and cells and in an unbiased manner at the whole genome to really be sure about this. Still their finding of no detectable off-targets so far is impressive.


2. Indels? The Indels present at the sasaran locus even under ideal circumstances in just under 30% of embryos are a big deal and remain a major problem. See part of Figure 3A above in the experiment where they found the 27% of embryos having Indels.


3. Clinical intent overall and NAS report on human gene editing? These folks make no bones about their hope to one day use this kind of technology for human reproduction with specific clinical goals. While they also included some appropriately cautionary statements about future clinical use, at the same time some language such as envisioned possible “rescue” of embryos was potentially concerning. I am highly skeptical that gene editing in the human germline can make sense as a safe and more effective approach than embryo screening by PGD and PGS.


4. Will this paper embolden others to dive in to this space too? Perhaps it will catalyze more research on CRISPR in human embryos. That could be both good in the sense of learning more, but also risky in terms of not everyone doing such a good job as these authors did in considering ethical implications and even in the technological level they used. Also, where will everyone get eggs and sperm for studies?


5. Will this paper lead to a negative, perhaps political reaction? I do think it is better than 50-50 that there will be some kind of political reaction from conservatives about this development and possibly some kind of proposed restrictions.

And more questions pop up now that I’ve read the paper.

10,000 eggs or embryos? What if to get to a clear answer on whether this technology is safe and effective it takes 1,000 or 10,000 human embryos, and hence eggs? CRISPR’ing human embryos at that scale might be needed to get clearer answers on efficacy and safety. Does the hypothetical potential benefit of pursuing human germline editing justify that? These are not every day run of the mill cells. Procurement and use of human eggs and embryos requires extra consideration.

What about epigenetics? Does CRISPR’ing human embryos lead to epigenetic effects that have biological outcomes, some of which may be negative?

Flying blind OK? Another thing to keep in mind is that if this technology were taken in a reproductive direction, you could not analyze the embryos in depth like they did in this paper. You’d have to largely fly blind. At best you could pluck a few blastomeres off for analysis, but you have to leave most of the embryo behind to actually get a baby.

Overall, this is an impressive paper, but one that also raises the stakes on future CRISPR use in humans.

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√ 4 Key Reasons Mitalipov Paper Doesn’T Herald Safe Crispr Human Genetic Modification

We can be confident that human genetic modification via CRISPR’ing of embryos soon will be safe and effective after that new exciting Mitalipov team paper, right?

Wrong.

The reality is far more complicated and interesting on the tech side.

In a nutshell, I see the paper as a significant scientific, but not necessarily medical advance.

The media coverage overall has painted too rosy and simplistic a picture of the Mitalipov paper. Setting aside tough bioethical and societal issues regarding human genetic modification for the moment in this post, there are 4 major, mostly overlooked technical reasons why the Mitalipov CRISPR human embryo paper is unlikely to herald safe human genetic modification.

1. I dream of Gene-y. The “best” disease-associated gene to sasaran with CRISPR in humans would be one that CRISPR always “hits” perfectly. The Mitalipov team probably gave deep thought to picking the gene they did for CRISPR targeting. It’s a gene going by the name MYBPC3 that is associated with a fatal type of heart defect called familial hypertrophic cardiomyopathy (HCM). That disease association is an important reason for trying CRISPR on the mutation in this gene. However, most likely at least one big technical reason they chose MYBPC3 was because it had so few predicted possible CRISPR off-target sites (meaning other places in the genome found by computer algorithms that CRISPR might stray to and make damage). If I’m right about this, then they were just being smart in that choice and I would have probably done the same thing in their shoes. But the targeting of this hand-picked gene means that the upbeat findings on accuracy with reverting mutant MYBPC3 are probably unlikely to be representative of efforts to “gene edit” disease-causing mutations more generally. By analogy would you like to throw a dart at a dartboard where the bull’s-eye takes up fully two-thirds of the dartboard (MYBPC3?) or where the bull’s-eye is just one thirtieth (some other disease-causing mutations)? For many diseases you may in effect have no choice but to go for the far tougher bull’s-eye because of the nature of the particular gene and its disease-associated mutation.

Isn’t it possible for all major disease-associated gene mutations that CRISPR will work as well (or even better) than MYBPC3? Nope, that’s not the way the real world works unfortunately.

In fact, one of the authors (Jin-Soo Kim of the Institute for Basic Science in Daejeon, South Korea) specifically emphasized to Nature News the low predicted off-target rate of this gene:

“Even so, Kim notes that the CRISPR–Cas9 error rate can vary depending on which DNA sequence is being targeted. The MYBPC3 mutation, in particular, was predicted to produce relatively few opportunities for off-target cutting.”

It is also possible the team picked MYBPC3 because its mutation is very small (only 4 base pairs and in theory easier to repair) or they had the human sperm donor lined up with this mutation.

A combination of factors most likely guided the team in gene choice.

Other gene mutations are going to be far tougher because they will be prone to dramatically more off-targets (see below) and perhaps more Indels (see below). Many mutations are large and complicated as well.

2. Off targets there, but not oft found? There’s also the likely possibility that the team unintentionally missed finding some off sasaran effects of CRISPR that were in their modified embryos, but not found because of how they did the sequencing. The very next quote in that Nature News piece is from Keith Joung on this concern:

“Just because the team did not find off-target changes does not mean that the changes aren’t there, cautions Keith Joung, who studies gene editing at the Massachusetts General Hospital in Boston. “Although this is likely the widest examination of off-target effects in genome-edited human embryos performed to date,” he says, “these investigators would need to do much more work if they wanted to define with certainty whether off-target effects do or do not occur in this context.”

Give the Mitalipov team credit for the screening they did do, which was relatively a lot, but much more is needed to be even close to sure about this, especially if one has clinical hopes as this team does.

3. Indel pain in the neck. The metaphor behind the language and concept of “gene editing”, the preferable phrase to “genetic modification” within the scientific politics of today (admittedly I sometimes use this phrase myself), suggests precision changes as do other metaphors like “genome surgery”. However, even if CRISPR-Cas9 avoids off-targets and sticks to the gene of interest, it can often make these things called “Indels” short for insertions and deletions right in sweet spots in genes. Indels often functionally kill genes entirely rather than precisely changing them. The Mitalipov team found Indels more than 1/4 of the time in embryos even under their most optimized conditions. The rate of Indels needs to be at or very close to zero to begin to have any reasonable chance of clinical safety of using CRISPR in the human germline. Plus, at other mutant genes that may be targeted in human embryos, Indels may be much more commonly created by CRISPR than at MYBPC3. We just don’t know.

4. Mosaic monitor. Mosaicism with CRISPR is where the cells of the embryos after introduction of CRISPR-Cas9 machinery don’t all have the same genome any more. For example, some cells in the same embryo may be normal and some mutant. There’s a genomic gemisch. That’s generally not good for ultimate health so mosaicism would be unsafe for hypothetical clinical applications of CRISPR in humans. One of the most impressive things about the Mitalipov CRISPR embryo paper was that they reportedly got rid of most (just 1 mosaic found) mosaicism in CRISPR’d human embryos. However, this was essentially just a very narrow test case study with one male sperm donor and one or a few women who donated eggs. Thus, the embryos used were very similar. More broadly, there is likely to be substantial variability in propensity to embryo mosaicism in part related to the unknown characteristics of specific gamete donors.

PGD reminder. Beyond the technical challenges, the fact is that almost anything CRISPR could do of medical use heritably in humans is already achievable using embryo screening including by the common, proven method called PGD. Think of it this way by analogy. Let’s say you have 8 books with 4 having errors and 4 not having errors. You have a very reliable way to know which books are which, and you only need 1 correct book. Do you try to correct the 4 errant books knowing that you could easily make more errors yourself in trying to fix the error, or just pick from one of the easily identifiable perfect ones?

Bottom line. For all these reasons, we should all be more cautious in making meaning from this one paper. There’s a long tough road ahead with a marathon of challenges (and the authors rightly acknowledged many of these so kudos to them) if one has clinical aspirations for CRISPR in the human germline.

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√ 1St Knockout Human Embryos Made With Crispr: My Take On The Pub

Scientist make knockout human embryos with CRISPR?

Today we see a new Nature paper (Fogarty, et al.) on CRISPR “gene editing” of human embryos, this time from the UK from Kathy Niakan’s group.

Niakan got UK permission about 18 months ago to CRISPR healthy human embryos so they’ve been hard at work since. Because Fredrik Lanner of Sweden (see my interview here) also has governmental permission to CRISPR healthy human embryos, I’m guessing we’ll see a paper from his lab soon too. If I had to predict I would bet that their paper will also induced targeting of OCT4, but perhaps also with another key pluripotency gene being CRISPR’d as well such as NANOG.

What’s the scoop on this Fogarty, et al. paper from Niakan’s team?

How does it relate to the Mitalipov group Ma, et al. paper that has stirred so much debate?

This new paper is entitled, “Genome editing reveals a role for OCT4 in human embryogenesis”. It comes shortly after the big Mitalipov group Ma, et al. paper also in the same journal that has left many wondering in that case if CRISPR gene correction even happened.

In the new Fogarty paper, the team introduced CRISPR-Cas9 into human zygotes to sasaran the key pluripotent gene OCT4/POU5F1. They injected a sgRNA–Cas9 ribonucleoprotein complex for the targeting and did so in S phase embryos approximately 5 hours prior to cytokinesis. That’s a fairly late beginning to gene targeting in terms of trying to prevent mosaicism and in fact they mention that a subgroup of embryos were at a later stage at thawing and so gene editing likely occurred developmentally later in those. In both cases it appears gene editing likely happened during mitosis, not earlier.

The Niakan group team found that they could efficiently target OCT4/POU5F1 in this way for disruption creating knockout human embryos that lack expression of that gene. In turn they report that loss of OCT4/POU5F1 leads to early developmental problems for the human embryo, somewhat earlier than in what happens in OCT4/POU5F1 knockout mice.

One of the first things that jumps to mind is that OCT4/POU5F1 (Oct4/Pou5f1 in mice) has been studied a ton in cultured stem cells, reprogramming and in mouse and other embryos. For this reason, as I was reading the Fogarty paper I kept being sure to ask myself, “What’s clearly new here besides the human context?” The earlier human phenotype is one reported difference and they also found evidence of OCT4 function beyond strictly pluripotent cells of the ICM.

They did the CRISPR-Cas9 targeting of OCT4/POU5F1 first in human ES cells and found interestingly that they could predict from the ES cell context many of the Indels that they found in the human embryos by sequencing. Notably, they also report an absence of detectable off-target sites, which is encouraging. Off targets may still be there, but not be detected due to limitations on sequencing of individual cells in human embryos. Still again this apparent accuracy is a positive sign that the technology can have good accuracy.

The Fogarty, et al. team found that the control method of injection gene editing machinery without guides (no genetic change) still led about 1/2 of embryos to fail to develop, but that this is roughly the same rate observed in published studies for unperturbed human embryos. In other words, about 1/2 of human embryos fail even without any poking or prodding in the lab. The CRISPR-Cas9 gene targeted embryos largely lacking OCT4 had a far lower rate of development though.

The Fogarty team reported that loss of OCT4/POU5F1 led to a number of important transcriptomic changes including many that one would expect given what is already known about pluripotency regulatory machinery. For instance, they found that knockout of OCT4/POU5F1 in the human embryos led to increased expression of some differentiation-associated genes. In addition, they report more heterogeneity in cellular gene expression in the knockouts, suggesting early embryo cells kind of lose their way and controlled cellular identity without OCT4.

How is Fogarty different than the Ma paper from Mitalipov?

First, in the Fogarty paper, the team used leftover embryos from preoccuring IVF procedures, whereas the Ma, et al. team made embryos themselves via IVF expressly for use in research. This is a very important distinction at a bioethics level. So the starting materials had a different origin.

Second, in Fogarty, the goal was to disrupt a gene via Indels, whereas in Ma the goal was to repair a mutant gene, which is a huge difference. In Ma, Indels were to be avoided. In Fogarty no repair templates were introduced because again generation of Indels was the only goal so it is difficult to compare results in some ways to the Ma paper.

Third, on a higher level, the intent of the research in these papers is also different in that Mitalipov clearly says he hopes this technology can be used in a reproductive, heritable fashion in humans, whereas Fogarty, et al. are primarily saying their work is about advancing knowledge, although they too mention potential clinically-relevant outcomes in the future.

Fourth, it is interesting that Fogarty did not report any evidence of inter-homologue repair, which is the hot button issue in the crosshairs related to the Ma paper. But Fogarty also didn’t seem to find very large Indels of the kind that would have made detection of Indels in the Ma paper definitely fail to work, as had been put out there as a possible alternative explanation for the Ma claim of inter-homologue repair. However, the nature of Indels will vary by gene so the jury’s out.

In addition, finally Fogarty reported plenty of mosaicism, whereas the Ma paper made a point of how little (only one mosaic embryo) that was detected. This difference may be due to technical distinctions between the studies.

Overall, this new paper is interesting and I expect more such papers are coming. We need to evaluate all such papers where there is use of CRISPR in human embryos both at scientific as well as ethical and policy levels.

In the abstract of the Fogarty paper, it’s striking that their main conclusion doesn’t even mention OCT4: “We conclude that CRISPR–Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.” In a sense, this paper is more about CRISPR use in human embryos than about OCT4.

I have a feeling in a few years it’ll seem less of a big deal and such papers will not routinely be in journals like Nature. Maybe it’ll go through phases as it did with IPS cells. In the very beginning many IPS cell papers were in top journals, but then people got kinda used to it. The buzz wore off.

Still now it’s not everyday that a lab uses to CRISPR to genetically modifies human embryos. Yet.

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√ Wearing ‘Good Genes’: Trump Eugenics

Is President Donald Trump a fan of eugenics? He seems to believe that he comes from greatly superior genetic stock.

He hasn’t said much of anything about CRISPR ‘gene editing’ technology that can readily alter the genetic code of cells of nearly any organism, including humans, but Trump has a fascination with the concept of “good genes” that sounds eerily similar to eugenics and could link together CRISPR & Eugenics.

If you watch the video above, you’ll see many striking quotes from Trump on his “amazing” genes.

What’s the deal? Could Trump be a eugenicist without even knowing it? Where’s the line if any between snobbery and eugenics? Can the idea of genes unite the two?

The word ‘eugenics’ can be taken literally to mean good birth or good genes. In the past, eugenics as a movement has largely been associated with societal disasters where certain individuals or groups decided that some segments of society were intrinsically inferior. As a result, these groups were oppressed or even killed. The Nazis embraced eugenics, but so did many Americans especially in the early 20th century when forced sterilization was not so unusual leading to tragedy.

It’s easy to find odd quotes from Trump about topics related to “good genes” that ring a eugenics kind of bell. For instance, just Google, “trump good smart genes” as a search term and watch the results containing his quotes pile up. Perhaps not surprisingly, Trump often brags about his family’s superior genetic stock and even has made remarks of a similar kind about members of his cabinet.

From Newsweek:

When talking about his granddaughter Arabella Kushner, “”She’s unbelievable, huh?” Trump said. “Good, smart genes.”

“I consider my health, stamina and strength one of my greatest assets,” Trump tweeted in December 2015. “The world has watched me for many years and can so testify—great genes!”

“Dr. John Trump, uncle, for many years at M.I.T.,” he also wrote in May 2013. “Good genes, I get it!”

From the president’s biographer Michael D’Antonio last year.

“The [Trump] family subscribes to a racehorse theory of human development,” D’Antonio said in his PBS documentary, The Choice. “They believe that there are superior people and that if you put together the genes of a superior woman and a superior man, you get a superior offspring.”

This sounds eugenic to me and again that YouTube video above of Trump quotes is pretty wild. A couple of weeks ago Time magazine had a piece on Trump and eugenics that started out, “President Trump brags a lot about his genes.”

What does it mean if your country’s president believes in eugenics and that he is superior to pretty much everyone else due to his genetic makeup? I don’t think it’s helpful to put it mildly. The potential connections between Trump and the white supremacy movement resonates here too on a eugenic level.

Some people apparently believe in “better living and greater intelligence” through genetic modification including potentially via CRISPR use in the human germline. Maybe Trump thinks he and his uber-family wouldn’t need such interventions. If one assumes for argument’s sake that Trump is a pretty smart guy in a basic sense, it still doesn’t mean he’s ‘better’ than anyone else. Trump’s apparent great lack of empathy for others, overconfidence, and many other traits aren’t exactly positive to society in my opinion.

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√ Countering That Pro-Heritable Human Crispr Wsj Piece

It’s germline CRISPR time, right?

Wrong.

But the particularly enthusiastic supporters of heritable human CRISPR often cite hypothetical benefits in glowing terms, but either don’t mention risks or strongly downplay them. These fans also tend to leave alternative, proven and safe technologies such as preimplantation genetic diagnosis (PGD) out of the discussion or only mention them as an afterthought. In reality, the vast majority of anything that CRISPR could hypothetically achieve heritably in human reproduction can be done better, more simply, and at dramatically lower risk by PGD.

A new WSJ opinion piece from Henry I. Miller of Stanford enthusiastically espouses the pro-human germline CRISPR view. Note, Miller also wrote an earlier pro-human germline CRISPR piece entitled, “In defense of germline gene therapy” that has stirred some controversy. I have a very different perspective than Miller.

Amongst other things Miller in the new piece argues that now is probably the time for trying to make babies with CRISPR in part because of that recent Mitalipov Nature Ma, et al. paper:

“Now, after the Oregon study, the technology is arguably at the stage where clinical trials could be undertaken to see whether gene-edited human embryos can develop into healthy babies.”

There are some problems with this statement.

First of all, what about those serious, unresolved scientific doubts on the Ma paper? The manuscript’s main conclusions remain up in the air, but even if they are ultimately proven 100% correct, do we base high-risk, new clinical science on one paper targeting one mutation conferred to embryos by one sperm donor in research done by one research group? We shouldn’t. It’s unwise to rush into human clinical research of any kind and that’s especially true if it includes heritable genetic modification. You first need reproducible, rigorous data to back you up from sufficiently power studies and a good sense of anticipated risks.

Are we there yet? No. Not even close.

Secondly, what if the answer to that implied experimental question in the above quote from Miller’s piece “to see if embryos can develop into healthy babies” is “no”? It could be disastrous. For instance, what happens to the unhealthy or deceased CRISPR’d humans you made in your experiment that didn’t turn out the way you had hoped? We just say “oops”? Or what if the babies seem OK at first, but then later they become ill or die?

These are not fun questions to ask or ponder, but they are deeply important if we are going into this with our eyes open. I wish Miller had discussed the risks of what he is proposing more in the WSJ piece, but that would have undermined his own argument. I realize that biomedical science needs to take on a certain level of risk to boost innovation, but the risks for heritable human CRISPR at this point would be sizable and span multiple generations.

To be clear, I’m not referring to innovative clinical trials of the non-heritable use of CRISPR in cell-gene therapies, which I strongly support. These too have risks, but have much more data behind them and do not involve heritable changes.

I emailed Miller to ask a few questions about his article. In response, he described the goal for his WSJ piece this way:

“The primary point of my article was the unwisdom of the absolute prohibition of clinical studies by federal law (FDA) and binding guidelines (NIH). If regulators at FDA or NIH feel that a tawaran is premature or inappropriate for any reason, they can reject it, but absolute prohibitions are bad public policy.”

However, in his piece he wasn’t so much arguing against a prohibition, but in favor of trying CRISPR in the human germline now. Keep in mind that even absent a clear prohibition, the sense I have is that the vast majority of scientists still don’t favor going ahead with germline CRISPR now.

While I have at times advocated for an at least temporary moratorium (established by the scientific community itself, not by law) on heritable use of CRISPR in humans, I realize that there are practical difficulties to that idea. Who if anyone enforces a moratorium and how? Where? What about the many different perspectives around the globe that may make a moratorium far from universal? There are no easy paths or fixes in this arena, but the scientific community needs to take stronger stands in my view that now is not the time to proceed and that time, if it comes, is a long way off.

Miller’s piece also resonated for me with a recent USA Today opinion piece by Alex Berezow of the American Council of Science and Health (ACSH) and Ben Locwin of the BioPharma Research Council. Their piece told us all to kind of ‘chill out’ about the idea of designer babies.

I emailed Alex to ask about Miller’s piece and also because some critics of Miller suggested in the past that he was affiliated with ACSH. Alex said that Miller isn’t part of ACSH. Alex also had this to say about his own views of human germline gene editing:

“My position is that we should proceed cautiously, keeping in mind that “designer babies” are science fiction. It’s easier to discard genetically defective embryos than to fix them. I see the biggest promise for CRISPR being in research, assuming the federal government eventually lifts the funding ban on deriving new stem cell lines from human embryos.”

It seems there’s common ground there between me and Alex on the wisdom of PGD-based embryo screening rather than attempting risky heritable human CRISPR. As to concerns (or not) over designer babies, much depends on how one defines them and what genes are targeted.

I also asked Miller by email about PGD as an established alternative to human germline genetic modification and he had this to say:

“PGD isn’t feasible in all situations, such as if the male prospective parent has two copies of a defective gene where the disease is transmitted as an autosomal dominant, or where two sickle-cell homozygotes (autosomal recessive) wish to have children.”

Yes, PGD isn’t a panacea (and it can have its own bioethical issues), but the instances where PGD won’t work (the unique window where CRISPR could in theory help, potentially justifying its weighty risks with heritable use) include only very rare and sometimes even hypothetical situations. The rarity of such reproductive cases doesn’t mean they should be dismissed entirely, but the context is important.

Arguably, Miller and others who are so upbeat about human heritable CRISPR should be mentioning the reality that situations where CRISPR could hypothetically potentially fill an important clinical gap left by existing technologies like PGD are realistically probably in the realm of one-in-a-million kind of cases. Even if we knew it to be relatively safe, there just is no convincing case now for a broad need for taking a stab at using CRISPR heritably in humans to try to prevent common genetic diseases rather than using PGD.

When pressed on this reality, some folks start mentioning other tangential hoped-for outcomes of reproductive human CRISPR use, such as supposedly preventing more IVF-produced human embryos from being discarded by “fixing” and trying to use them, but that’s a weak argument too. So, you are going to make, CRISPR, and analyze (and hence in a sense discard) potentially thousands of human embryos for research to try to figure out how to hopefully, safely correct mutations in some embryos in the future primarily so they might not be discarded after PGD earlier determined they have mutations? Isn’t there some inherent illogic there?

Furthermore, no matter how good the data get from human embryo CRISPR preclinical studies, there will always be some unique risk facing CRISPR’d human embryos used for reproduction. Again, that risk needs to be discussed realistically and balanced against a common-sense assessment of potential benefits compared to alternative technologies like PGD.

Miller in winding down his WSJ piece, makes a broad statement that I view as having some issues:

“It’s easy to invoke hypothetical fears when actual lifesaving interventions are decades away…Today they aren’t—and desperate patients deserve access to whatever cures this technology may be able to provide.”

The reality is that human germline use of CRISPR (assuming we decide it’s a prudent thing to try) is in fact one or more decades away if to be done in a responsible manner, even if some bozo could technically try it tomorrow or could have tried it last year. Also, the use of the word “cures” by Miller here is off-base because germline CRISPR would prevent, not cure disease. In addition, there is near zero basis at this point to argue that CRISPR can be specifically “lifesaving”. Overall, for these reasons this passage strikes me as hype.

In the end, heritable human CRISPR is still largely a wild idea whose time has definitely not come today. From scientific and medical as well as perspectives, it’s not something close to being ready to even just test reproductively in humans in a responsible way. Unfortunately, again it could be tried in an irresponsible manner at just about any time now.

Let’s keep the dialogue going on the many possible human uses of CRISPR including heritable human CRISPR. I realize that this discussion is a marathon even as CRISPR seems technologically like a sprint, but we have to keep talking things through to make the best decisions as a community.

For more on my “big picture” level perspectives and concerns related to the ongoing CRISPR research in human embryos see here with some key points regarding Mitalipov’s recent work, where I try to place it in a wider context and ask bioethical questions. Also, see my interview with Steven Pinker and my follow-up piece on why I largely disagree with him on this issue. You can also check out my book GMO Sapiens on possible heritable human use of CRISPR here.

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√ Top 7 Tech Hurdles To Human Germline Crispr

Human germline CRISPR raises major bioethical considerations, but what about technical issues?

Setting aside the many ethical issue about the general idea of human modification itself, could this really work? Yes in theory it could, but there are some very tough technological challenges that could and likely would cause failures or unacceptable outcomes at many steps along the way. These failures or unacceptable outcomes could easily involve real, live people who could be harmed or die. It’s very different than simple in vitro research so the tech side of it has been incredibly refined and robust. That’s not going to be easy.

What are the biggest technological roadblocks to responsible, successful (meaning safe and effective) use of CRISPR-Cas9 for heritable human genetic modification?

I see at least seven biggies.

This post is in part inspired by a section of my book on potential human use of CRISPR called GMO Sapiens.

1. Starting from point ACGT? Unknown initial genomic sequence of embryos

Let’s say we have a father-to-be, Mr. A, with a disease-causing mutation and a spouse Mrs. A who does not have that mutation. Dr. T, the leader of the scientific/medical team, gathers normal eggs from Mrs. A and then the sperm from Mr. A. Half of the sperm will be mutant and half will be the normal “wildtype” so the same will be true of the embryos that result from the IVF. Dr. T’s intention is to use CRISPR-Cas9 to revert the mutant allele back to wildtype in the embryos.

Dr. T would need to know the whole genome sequence of the embryos that the team will be trying to gene target. Here we have a Catch-22 situation. Dr. T’s team needs to know the starting genomic sequencing of the embryos before the intervention to design the CRISPR system components including the repair template with 100% confidence (although the very high level of sequence similarity between humans could allow for use of the so-called “reference” human genome for template design) and later to screen for off-target effects, but how does Dr. T get that information without destroying the one-cell embryos? She can’t.

So perhaps she settles for knowing Mr. and Mrs. A’s individual genomic sequences and then infers what the embryo/fetal sequence might be like? For example, for CRISPR guide RNA and repair template design Dr. T might assume that the mutated gene sequence in question will be from Mr. A in some embryos. They can also bring the reference human genome into play too.

It definitely feels a bit like flying blind. The best-case scenario would be that other than the mutation in question, the parents-to-be have exactly the same sequence in the overall region of interest.

2. Mutating wildtype normal embryos

Because the vast majority of possible clinical human embryo editing scenarios involve one parent with a heterozygous mutation and the other without that mutation like Mr. and Mrs. A, about half of their IVF-created embryos will have no mutation. So, Dr. T cannot know if she is attempting to edit WT or mutant embryos prior to CRISPR-Cas9 injection. Inevitably then they will be CRISPR’ing some normal embryos and probably mutating a subset of those via Indel creation. Is it permissible to genetically modify formerly WT human embryos that they intend to implant in a surrogate mother, potentially creating new disease-causing mutations in the worst-case scenario? I don’t think so. I don’t see how this could be avoided in this scenario.

Keep in mind again that if you sequence a few embryonic cells by PGD (more on that below) later during embryogenesis, a WT sequence showing up at the gene of interest could mean (A) this started out as a WT embryo or (B) it started out as a “mutant” embryo and you successfully corrected the gene mutation. How can you tell the difference at this level?

Let’s say that Dr. T encodes some clever, tiny “bar code” signpost via the CRISPR gene editing beyond the mutation correction to be an indicator during sequencing of a successful gene edit (rather than just a pre-existing WT allele). This could differentiate between correction or just starting with a WT embryo. However, then could the indicator–say something as small as 2 non-codon changing basepair changes–could cause trouble? Who knows?

3. Off-target effects

CRISPR-Cas9 is great and getting better (and we even know have an expanded toolbox with the ‘base editor’ chemical modification tech), but they aren’t perfect. There will always be a risk of it making edits in the wrong place in addition to a chance of making wrong edits such as Indels in the right place. Since Dr. T does not know the embryo’s starting genomic sequences, how does she know if CRISPR-Cas9 created an off-target mutation (e.g. as little as a single bp change that was undesired) or if instead that detected unexpected DNA sequence was just the unique sequence of the embryo to start with at some random place in the genome? There are a lot of naturally occurring variants including SNPs. Again, Dr. T would need to go back to Mr. and Mrs. A.’s sequences and hope she can get some clarity there along with the reference human genome. However, it is easy to imagine scenarios where the team just couldn’t be sure if a different say between a G and T at one place in the billions of genomic basepairs was a variant or an off-target effect.

Another practical confounding issue here is that Dr. T’s team is working with human embryos with few cells to use for screening. Ideally they need to screen for off-target effects so they need to be able to do whole genome sequencing (WGS). Can they accurately and reproducibly do WGS from only 1 or 2 embryonic cells (blastomeres) with accuracy down to the single basepair resolution across the entire genome? Of course, one could wait and get more cells from a fetus later after implanting the embryo, but then if problems arise we are talking about the possibility of an abortion coming into play.

4. PGD would usually miss mosaicism

To do the kinds of sequencing mentioned earlier, Dr. T is going to definitely do PGD and this will also be where she looks for mosaicism (i.e. some of the embryo’s cells have gene edits, while other cells in the same embryo do not and also there is the possibility of mosaic off-target effects). The team needs to know if there’s mosaicism and presumably if there is then they would halt the process as they do not want to create substantially mosaic humans who could have serious health consequences as a result.

Unfortunately if you intend to do full genome sequencing by PGD at the 8-cell stage you are relying on just one or two cells (or perhaps a few more if you do PGD at the blastocyst stage) to be predictive of the whole embryo. PGD of just one or a few cells could give you an entirely wrong view of the genotype of the other cells in the gene edited embryo. You might well incorrectly believe there is no mosaicism when in fact there is variability amongst the cells of the embryo that you just failed to detect. Again by PGD you should also be looking for off-target effects and you don’t want to limit that search only to one or a handful of cells either. PGD is crucial, but only a partial snapshot.

Another hurdle discussed in a previous blog post is coming up with a logical reason for using CRISPR in a clinical way in humans that would make it better than simply using already existing PGD technology by itself (without gene editing) to screen for embryos not possessing an inherited mutation. Why would Mrs. and Mr. A and Dr. T even want to try gene editing instead of using PGD all by itself? It’s difficult to come up with many scenarios where PGD alone wouldn’t work and where gene editing would be substantially better. An excellent Nature Biotechnology piece covers this question of when human modification might make sense. For instance, I thought Robin Lovell-Badge’s response was very cogent. One example is the rare situation with a parent-to-be who is homozygous for a dominant mutation or if both parents have disease-causing mutations.

5. The use of thousands of human embryo for optimization

The old saying goes that “practice makes perfect” and that kind of sentiment applies to CRISPR’ing human embryos even if it can never be 100% guaranteed to be perfect. Most attempts at human embryo editing in the lab are still likely to be informative so a knowledge base will build over time and improve the gene editing technology and methods. It will probably take many thousands of human embryos to optimize the system collectively and every specific lab doing a distinct kind of gene editing may require hundreds of embryos for its own optimization.

Is it acceptable to do such massive scale viable embryo editing simply for advancing knowledge? Also, where are you going to get all these eggs and embryos? I’m a supporter of human embryonic stem cell (hESC) research and embryos remaining from IVF procedures are used to make hESC (or are otherwise generally discarded), but not a tremendously huge number. In contrast, hypothetical yearly use of thousands of potentially newly generated human embryos simply to optimize gene editing and/or for advancing knowledge could start to get ethically and practically complicated. See my recent piece on the Mitalipov lab apparently already making and using hundreds of human embryos for CRISPR.

6. Trapped in a choice of “lesser evils” post-implantation? 

Let’s say somehow Dr. T successfully gets further along and the team has got a pregnancy with a genetically modified human embryo. If a duduk kasus then arises, what can they do about it? The team involved in this work could well find themselves trapped with a dilemma as to how to handle an adverse situation. The only options might both be problematic: (1) continuing a risky pregnancy of a human embryo/fetus with CRISPR-introduced genetic errors or (2) abort the pregnancy. If mistakes are relatively common in such clinical CRISPR research, is it OK to routinely abort such fetuses if problems arise?

Finally, what if health issues become apparent in Mr. and Mrs. A’s gene edited children only much later on down the road?

7. Unintended consequences.

The genome is a complicated jungle so even if you make the “right” edit with no off-target effects, how do you know you’ll get just the narrowly focused outcome you want?

Possible solution to some problems: gene editing in germ or stem cells?

The above discussion assumes a focus on gene editing conducted in one-cell embryos, but it is also in principle possible to gene edit mutations in germ cells. For example, one might do CRISPR in oocytes or even primordial germ cells (assuming successful working out some of the kinks in producing such sperm and egg-producing cells safely in humans), validate gene correction and lack of off-target effects in the cells prior to fertilization, and then proceed with IVF, implantation, etc. with the gene editing now in the rear-view mirror so to speak. This could resolve some of the issues mentioned earlier. At the same time this approach may have issues of its own such as the risks associated with prolonged manipulations of germ cells in the dish in the lab. It is also possible that the use of cultured primordial germ cells would pose unique risks as the cells change their epigenomes during their growth and manipulation in the lab. Still, this kind of approach is another, interesting option.

Bottom line. Overall to me the big picture at this time at least is one of serious technical hurdles in the way of responsible possible future clinical human genetic modification. Technology will improve and we may come to see solutions to some of these problems, but it seems unlikely that all these issues can be resolved completely. Throw in the numerous thorny ethical and legal issues and it seems even more difficult to imagine a future where there could be responsible, safe human germline genetic modification done with a unique, beneficial purpose. Despite all of this, I do believe that some people will go ahead and try making genetically modified people anyway.

Any responsible discussion of possible heritable human genetic modification needs to include dialogue on these kinds of technical hurdles and problems. When someone is aspirational about CRISPR germline use, it is worth asking them about these sorts of hurdles and also about what specific positive use they had in mind for heritable human gene editing that transcends what embryo screening can already achieve.

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