August 2, 2017

expert reaction to genome editing a disease-causing mutation in human embryos

A genetic mutation implicated in hypertrophic cardiomyopathy – a common heritable heart condition – has been corrected in preimplantation human embryos using the CRISPR/Cas9 genome editing technique, as reported by scientists in Nature.

Prof. Jeremy Pearson, Associate Medical Director at the British Heart Foundation, said:

“Every week in the UK 12 people under the age of 35 die of a sudden cardiac arrest and hypertrophic cardiomyopathy is the leading cause.  Our research has helped to discover some of the genetic defects responsible, but there is no way of curing the condition or stopping it from developing.

“This cutting-edge science shows that it is possible to correct an HCM mutation carried by the father’s sperm in a human embryo. Longer term, this could set the stage for new treatments to stop deadly conditions like HCM being passed down through generations.  However, it is far from being a clinical reality – more research is needed to understand whether the method is widely applicable, and there are significant ethical and legal concerns to be resolved before it could become lawful.

“Right now, we need better screening for the disease, and the BHF with the support of the Miles Frost Fund is working towards the national rollout of genetic testing for all families affected across the UK.”

Statement from the HFEA (Human Fertilisation and Embryology Authority), said:

“UK researchers can apply for a licence to edit human embryos in research, but offering it as a treatment is currently illegal. Introducing new, controversial techniques is not just about developing the science – gene editing would need to offer new options to couples at risk of having a child with a genetic disease, beyond current treatments like embryo testing. Our experience of introducing mitochondrial donation in the UK shows that high quality public discussion about the ethics of new treatments, expert scientific advice and a robust regulatory system are crucial when considering new treatments of this kind.”

Prof. Joyce Harper, Embryology, IVF and Reproductive Genetics Group, Institute for Women’s Health, UCL, member of the Nuffield Council on Bioethics working party on genome editing, and science advisor to the Human Fertilisation and Embryology Scientific and Clinical Advances Advisory Committee, said:

“Today an international group of scientists have published a detailed paper of their study on human germline genome editing. The work aimed to correct a mutation in the gene MYBPC3 which accounts for approximately 40% of the myocardial disease hypertrophic cardiomyopathy (HCM).  After using CRISPR-Cas9 to correct iPSC cells produced from a male patient carrying the MYBPC3 mutation, they went on to successfully edit zygotes and oocytes fertilised with the male patient’s sperm. The most promising results were obtained using oocytes.

“Most countries are debating the clinical, ethical and social significance of being able to genetically modify human embryos. In February this year, the National Academy of Sciences in the USA produced a 300 page document titled: Human Genome Editing: Science, Ethics, and Governance. The Nuffield Council on Bioethics working group in the UK are preparing a report which will discuss the ethical and social issues and will make recommendations relating to policy and practice. It may be that some countries never permit germline genome editing because of moral and ethical concerns. If the law in the UK was changed to allow genome editing, it would be highly regulated by the Human Fertilisation and Embryology Authority, as is PGD, to ensure it is only used for medical reasons.”

Dr Jim Smith, Director of Science, Wellcome, said:

“The use of genome editing in basic research will improve our understanding of how genes work and will help prevent and treat disease. This work overcomes some of the technical difficulties in editing the genome of the early human embryo, paving the way for future work.  We are still a very long way from contemplating the use of this technology in the clinic.  As the science continues to advance, it’s important that society should debate the use of genome editing technologies.”

Dr Helen O’Neill, Programme Director of Reproductive Science and Women’s Health, Embryology, IVF and Reproductive Genetics Group, UCL said:

“This study aims to address not only disease correction in an embryo but the numerous technical elements to consider when carrying out CRISPR genome editing: method of repair, means of delivery and the timing of this delivery. All of which result in different outcomes.  Ma et al used a multi-step approach indeed using alternative CRISPR constructs, repair templates and delivery methods carried out at different stages, to correct a genetic mutation in MYBPC3, which results in myocardial disease. The efficacy of the CRISPR complex was first tested in cells from patients, including means of testing the method of correction, before being used on human embryos. The affected patients’ sperm was then used to fertilize eggs, and following fertilization, the zygote (the earliest stage of an embryo) was injected with CRISPR machinery. This resulted in mosaic embryos, meaning only some cells of the embryo had been corrected, but not all.

“The next approach was to inject the sperm and the CRISPR machinery together into the eggs using intracytoplasmic sperm injection, a technique commonly used in IVF. This resulted in far more non-mosaic (corrected) embryos.

“The two major concerns of using CRISPR to edit an embryo are mosaicism and unwanted edits (referred to as ‘off-target effects’). This paper aims to address both of these issues and quantifies the level of each.  Thorough genome and exome sequencing revealed that the level of CRISPR mediated off target editing was low.

“The results reflect the much-needed progress in utilising human eggs and sperm in research for the prevention of diseases which have no current cure or treatment.  The technology is still in its infancy in terms of true clinical application, but the authors have acknowledged and attempted to address the current technical limitations of germline genome editing.”

Prof. Karen Yeung, Chair of the Nuffield Council on Bioethics inquiry into genome editing in human reproduction, said:

“The authors appear to have identified a promising method that could correct a disease-causing mutation that could otherwise be passed to future generations.  Our report will be published in 2018 after a year-long inquiry.”

Note: The report due to be published in 2018 will consider the ethical questions relating these kinds of genome editing applications, the suitability of UK and international regulation and policies, and will make recommendations on them.

(commenting on the disease in question, hypertrophic cardiomyopathy) Prof Sian Harding, Professor of Cardiac Pharmacology, Imperial College London, and Director of the BHF Centre of Regenerative Medicine, said:

“Hypertrophic cardiomyopathies such as this are one of the most common mutations-related diseases in the population, with a prevalence of around one in 500 people.  They are not curable, but can be helped by surgery or some drugs.  However, one of the main problems is they can frequently remain undetected until death due to heart rhythm disturbances occurs.  They are an important cause of sudden cardiac death in young people, especially when playing sports.”

Dr Anna Middleton, Head of Society and Ethics Research Group at Wellcome Genome Campus Connecting Science, and Vice-Chair of the Association of Genetic Nurses and Counsellors, said:

“This paper explores the mechanism for repairing a single defective gene in a very early stage embryo and shows that it can be done with a level of precision.  The science is really elegant but it should be replicated extensively before any firm conclusions can be drawn about accuracy and safety of the approach.

“The science makes a significant contribution to understanding one mechanism for repairing DNA.  This should be celebrated for what it is – exciting research.  It is far too early to extrapolate this into any clinical application and indeed the implantation of edited human embryos is currently illegal in many countries including the UK.  However, learning how to repair DNA will be extraordinarily valuable in working out how to treat genetic disease in the future.”

(from our friends at Science Media Center Germany) Prof James Adjaye, Chair of Stem Cell Research and Regenerative Medicine, Heinrich Heine University, said:

“The design of this study is well thought through and executed, especially regarding the comparative use of patient-derived induced pluripotent stem cells (iPSCs) and the derived embryos after in vitro fertilization.  A major and unexpected observation was that it seems that the DNA-repair mechanism(s) in the early embryo differs from the operative in the iPS cells and maybe even somatic / adult cells.

“This is an important finding for basic research and also a necessary pre-requisite for correcting mutations at the oocyte or preimplantation embryo level.  However, the results are currently based on one single patient as the donor, so additional replicates / donors are needed to statistically validate this finding.

“In earlier studies on CRISPR-Cas9 editing in human preimplantation embryos the authors added the gene-editing components after fertilization in contrast to the current study.  The low levels of mosaicism observed in this study can be attributed to the fact that gene editing occurred before the first cell division took place.  Interestingly the gene-corrected preimplantation embryos developed in a similar manner to the control embryos, with 50% developing to the blastocyst stage which is the stage where the embryos have the potential to develop further into the fetus and placenta.

“Ma et al did not observe off target effects and this they suggest might be due to the fact that they – unlike other previous investigators – used purified recombinant Cas9 protein instead of plasmid DNA, which might have enhanced the specificity due to a more controlled level of Cas9 enzyme activity.

“All said and done, more experiments are needed to address these and other yet to emerge questions before CRISPR-Cas9 can be routinely implemented in therapy.

“Pre-implantation Genetic Diagnosis (PGD) still remains the favoured and standard approach in preventing the transmission of deleterious genetically inherited diseases to offspring.”

Prof. Daniel Brison, Scientific Director, Department of Reproductive Medicine, Central Manchester University Hospitals NHS Foundation Trust, and Honorary Professor of Clinical Embryology and Stem Cell Biology, University of Manchester, said:

“This new study from Shoukhrat Mitalipov and collaborators appears to be a major advance in human embryo gene editing which brings the technology a step closer to clinical application in correcting inherited disease.  The scientists used their expertise in oocyte sperm injection to introduce the CRISPR-Cas9 gene editing components at the time of fertilisation, exactly as ICSI is routinely performed during clinical IVF treatments for infertility.  This appears to have increased the success rate of the gene editing by reducing the amount of inaccurate repair, and has also revealed interesting insights into the mechanism of DNA repair in human oocytes.  Using gene editing during fertilisation in this way is a clever idea, especially as we know that human sperm naturally contain a high level of damaged DNA due to environmental and other exposures, which must be repaired by the oocyte as part of fertilisation during natural conception.  So it is not surprising that the human oocyte is good at repairing damaged DNA and that it has very precise mechanisms for doing this which might be different to that in other cells.

“However even though this is a significant technical advance, it is worth noting that the technology was not 100% successful, rather it increased the number of successfully repaired embryos from 50% which would have occurred naturally, to 74%.  For gene editing of human embryos to be used clinically will require extensive safety testing and careful consideration of ethical aspects.”

Prof. Robin Lovell-Badge, Group Leader, The Francis Crick Institute, said:

“The headlines from the ‘scoop’ of this paper last week were clearly wrong. The paper is not about designer babies. It is aimed at preventing the inheritance of dominant mutations, in this case one that confers a severe risk of early onset cardiac disease that is a common cause of sudden death in young athletes. They show that the methods may work, although not in the way that was anticipated.

“Sperm was obtained from a man heterozygous for the mutation in the MYBPC3 gene, which were used to fertilise normal eggs donated for research. By introducing the genome editing components along with the sperm, the mutation was corrected at high efficiencies in the 50% of embryos that should have inherited the mutation (much higher than the rate seen in cells in culture), and importantly, they managed to avoid mosaicism (in all but one embryo). However, rather than repairing the mutation with a DNA template introduced along with the Cas9 and guide RNAs, the repair mechanism used the maternal (the egg’s) copy of the gene. This was surprising, as it would be expected that the many copies of the DNA template would be used in preference to the single maternal copy.  This is not simply ‘mother knows best’, but it suggests that a novel mechanism of DNA repair operates in this very early stage of human embryo development.

“There is still much to be done to establish the safety of the methods, therefore they should not be adopted clinically. However, this mechanism of genome editing could in theory be harnessed to correct mutations coming from a father; as in the case described, to increase the chances of obtaining an embryo that does not carry a dominant disease-causing version of a gene. Alternatively, if the father was homozygous for such a mutation, it would allow him to have a genetically related unaffected child, which would otherwise not be possible. However, it seems to be a one-way repair process – it would probably not work if the mother were carrying the mutant gene (indeed, it might lead to embryos homozygous for the mutation). This will require new methods to permit efficient DNA template-directed repair.  The mechanism revealed in this paper would similarly not permit more sophisticated genetic alterations.  The possibility of producing designer babies, which is unjustified in any case, is now even further away.”

Further explanatory notes from Prof Robin Lovell-Badge:

Genome editing relies on normal cellular processes that are required to repair DNA whenever it is broken, which happens, for example, when DNA is replicated during cell division.  The genome editing components introduce a break in the DNA at a designated position in the genome.  Repair by ‘non-homologous end joining’, which simply sticks the broken ends together, is efficient, but it leads to small mutations, and can’t be used to correct a faulty gene.  Homology Directed Repair makes use of a DNA template which has some homology to the part of the genome being targeted, and it copies the DNA sequence of the template into the latter to repair the genetic defect.

Mosaicism is commonly seen when the genome editing occurs late, after the DNA has replicated in the egg or after the first cell division, such that the resulting embryos have a mixture of edited and unedited cells.  The latter needs to be avoided because it would not necessarily rescue any child born from the genetic defect if it was present in some of his or her cells.  However, it is also beneficial because it would allow the embryos to be tested by PGD to verify that the correct genetic alteration had been made.  With a mosaic embryo, it would be impossible to know if the biopsy reflects what has happened in the rest of the embryo.

Prof. Peter Braude, Emeritus Professor of Obstetrics and Gynaecology, King’s College London, said:

“This remarkable paper demonstrates just how rapidly the field of genome editing has progressed since the CRISPR-Cas9 system was voted science breakthrough of the year in 2015.  The substantial author list also shows the very best of international collaboration (USA, China and South Korea) in order to bring high-end genetic and cellular technology to bear on a technically difficult and controversial area of science and medicine.

“Whilst there are still some important potential hazards such as mosaicism and off-target effects, substantial progress has been made here on understanding how they might happen and be ameliorated.

“Preimplantation genetic diagnosis (PGD) with embryo selection is still the only current practical option for couples to prevent transmission of genetic disorders to their offspring, but this paper presents a possible future alternative especially in dominant disorders (like Marfan syndrome, Huntington disease or Hypertrophic Cardiomyopathy as in this paper), as the editing correction seems more reliable when there is one normal gene present as is usually the case in such inheritance.  Corrective editing could reduce the proportion of embryos discarded as being not suitable for transfer after PGD.

“Although use of this method clinically would not be allowed under current legislation in this country, with this paper the possibility of germline genome editing has moved from future fantasy to the world of possibility, and the debate about its use, outside of fears about the safety of the technology, needs to run to catch up.”

Dr Yalda Jamshidi, Reader in Genomic Medicine, St George’s, University of London, said:

“The study led by Shoukhrat Mitalipov of Oregon Health and Science University is the first to show successful and efficient correction of a disease-causing mutation in early stage human embryos with gene editing.

“Two recent studies by groups in China had attempted to modify genes using gene editing in human embryos, however they had difficulties in ensuring that all the cells in the resulting embryo contained the repair.  This new study is exciting because it shows the possibility to overcome this difficulty by injecting the repair enzyme and the mutation-carrying sperm into the egg at the same time.

“The scientists used healthy egg cells donated by women, and sperm of a man affected by Hypertrophic Cardiomyopathy, and unlike the previous two attempts showed that the gene editing technique can be very efficient.

“Just over 70% of embryos were corrected effectively, and using their modified technique none of the 42 corrected embryos contained unchanged cells.  People carrying a mutation have a 50% chance of passing it on to their own children.  Therefore, using the gene editing technique scientists were able to increase the probability of inheriting the healthy gene from 50% to just over 70%.

“Hypertrophic cardiomyopathy (HCM) is a common inherited condition affecting 1 in 500 people.  It can lead to heart failure and the sudden death of apparently healthy people.  In 40% of families with HCM it is caused by a single copy of a mutant MYBPC3 gene.  Currently couples affected by genetic diseases such as HCM can create embryos through in vitro fertilization, screen them in the lab and implant only ones free of the defect.  Using the gene-editing technique however would mean that couples would potentially require fewer IVF cycles to obtain a healthy embryo.

“The study also showed that using their modified gene-editing technique, no unintended mutations were observed.  This is another concern when using gene-editing tools in the embryo as both the intended and unintended changes could be passed on from generation to generation.

“Importantly gene-editing research in human embryos is subject to appropriate legal and ethical rules and oversight.  The embryos in the study were not allowed to develop for more than a few days and were never intended to be implanted into a womb – as would be the case if similar research were to be carried out in the UK.

“Families with genetic diseases have a strong drive to find cures.  Whilst we are just beginning to understand the complexity of genetic disease, gene-editing will likely become acceptable when its potential benefits, both to individuals and to the broader society, exceeds its risks.”

Prof. Darren Griffin, Professor of Genetics, University of Kent, said:

“Given the increasing success with CRISPR-Cas9, in many ways a report like this was inevitable.  The work appears to be convincing, scientifically sound and performed with great thoroughness.  It is nonetheless very challenging with a number of issues to consider.

“The first is clinical need.  Presumably the approach is mostly useful when preimplantation genetic diagnosis (PGD) indicates there are no embryos available for transfer.  This is unlikely to be a large number.

“The second is safety.  As the authors clearly point out, the safety of the approach (i.e. whether there are any unintended consequences to the health of the embryo and the subsequent baby) is of paramount concern.

“The third is practicality.  If the patients are to have a PGD to identify the embryos in need of ‘correction’, then the CRISPR-Cas9 treatment, then a second PGD to establish whether the procedure has worked, then one wonders whether the whole procedure would gain in popularity, even if it was licensed.

“Perhaps the biggest question, and probably the one that will be debated the most, is whether we should be physically altering the genes of an IVF embryo at all.  This is not a straightforward question, the academic disciplines of law, social policy, psychology and philosophy may all become involved.  Equally the debate on how morally acceptable it is not to act when we have the technology to prevent these life-threatening diseases must also come into play.”

Prof. Shirley Hodgson, Professor of Cancer Genetics, St George’s, University of London, said:

“This research is an important step forward in the use of CRISPR techniques to correct mutations in the early embryo.

“The novel findings of this work are the use of the technique at the first cell of the embryo, thus avoiding mosaicism, and it seems that used at this stage the repair appears to use the cell’s own repair mechanism and normal copy of the gene.

“However, the researchers have only corrected one specific gene, and this was only correctly replaced in 42 out of 58, and then two thirds of 19 embryos, so clearly further experiments are necessary to confirm these observations.  These researchers looked for off target mutations and did not detect any, but this remains an important concern in the use of this technique in man.

“These embryos would of course need to be tested to see which version of the gene was present, which is the procedure already in use for preimplantation genetic diagnosis, PGD, a well tried and tested way to prevent genetic diseases being passed on to the offspring of mutation carriers, with established ethical governance.  Each mutation that is to be corrected using this technique requires a different set of experiments to make the CRISPR technique appropriate for that gene, which is not necessary for PGD.

“An interesting problem encountered in this technique is the fact that the cell uses its own copy of the normal gene as a template for repair.  This indicates that this technique may not be applicable to autosomal recessive disease where the disease is caused when an individual has two copies of the faulty gene.

“Overall the researchers seem to have overcome the main problems encountered in the Chinese experiments (mosaicism and off target mutations) but it will require considerably more research on many more embryos and with many more genes before confidence in this technique can be established.  The ethical oversight of such research is becoming even more urgent as this methodology becomes increasingly applicable to human genetic disease.”

* ‘Correction of a pathogenic gene mutation in human embryos’ by Hong Ma et al. will be published in Nature on Wednesday 2 August 2017. 

Declared interests

Dr Jim Smith: “Jim is also a group leader at the Crick.”

Dr Helen O’Neill: “No conflicts / nothing to disclose.”

Prof. Sian Harding: “I have a collaborative project with Mitalipov on pluripotent stem cell-derived cardiomyocytes and we were together in a Foundation Leducq consortium grant.”

Dr Anna Middleton: “Member of the British Society of Genomic Medicine.”

Prof. Daniel Brison: “I supervise PhD students and receive academic grant funding from NIHR and MRC in the general area of fertility research including human embryo development.   I have no commercial interests.”

Prof. Robin Lovell-Badge: “Robin Lovell-Badge has no conflicts of interest.  He uses genome editing methods in his own research, but this does not involve human embryos.  He was a member of the NAS Study Committee that produced a Report on Human Genome Editing, of which one of the senior authors, Juan Carlos Izpisua Belmonte, also served.  However, they have not worked together subsequently and they have not collaborated on any aspects of this or other research.”

Prof Peter Braude: “I have no conflicts of interest to declare.”

Dr Yalda Jamshidi: “I have no declarations of interest.”

Prof. Darren Griffin: “Prof Griffin is the Director of the Centre for Interdisciplinary Studies of Reproduction (CISoR, http://www.kent.ac.uk/cisor) and is on the board of the Preimplantation Genetic Diagnosis International Society.”

Prof. Shirley Hodgson: “I have no conflicts of interest.”

None others received.