Nanoethics DOI 10.1007/s11569-016-0259-0

ORIGINAL PAPER

CRISPR/Cas9 genome editing – new and old ethical issues arising from a revolutionary technology Martina Baumann

Received: 9 December 2015 / Accepted: 1 April 2016 # Springer Science+Business Media Dordrecht 2016

Abstract Although germline editing has been the subject of debate ever since the 1980s, it tended to be based rather on speculative assumptions until April 2015, when CRISPR/Cas9 technology was used to modify human embryos for the first time. This article combines knowledge about the technical and scientific state of the art, economic considerations, the legal framework and aspects of clinical reality. A scenario will be elaborated as a means of identifying key ethical implications of CRISPR/Cas9 genome editing in humans and possible ways of dealing with them. Unlike most other discussions of CRISPR/Cas9 germline editing, which are generally based on deontological arguments, the focus in this case will be on a consequentialistic argument against certain applications of germline and somatic editing that takes not only the potential benefits and risks but also socioeconomic issues into consideration. The practical need for an indication catalogue, guidelines for clinical trials, and for funding of basic research will be pointed out. It will be argued that this need for regulatory action and discussion does not stem primarily from the fact that CRISPR/Cas9 germline editing is revolutionary in terms of its ethical implications and potential for human therapy, although this is the prevailing view in the current discussion. Understanding the value and interest dependency of arguments put forward by different stakeholders and learning from past

M. Baumann (*) Munich Center for Technology in Society, TU Munich, Arcisstrasse 21, 80333 Munich, Germany e-mail: [email protected]

debates related to similar technologies might prove a fruitful method of reaching judgments and decisions that come closer to a consensus upon which society as a whole can agree - which after all should be the true goal of an ethical debate and of bioethics. Keywords Crispr . Genome editing . Germline modification . Bioethics . ELSA

Introduction The development of the CRISPR/Cas9 genome modifying technique, which has recently been used in human embryos [1], was intended to herald in an Bera of straightforward genome editing^ [2] featuring great advances in science and medicine [3, 4]. CRISPR/Cas9 allows scientists to genetically Bedit^ the genome sequences of higher organisms from mice to monkeys with unprecedented ease and speed, high precision and lower costs than former genome modifying tools like TALENs1 and ZFNs2 [5, 6]. DNA sequences may be inserted, removed or changed at virtually any position in the genome [5]. In principle, several modifications can be performed simultaneously in one genome, which opens up the possibility of treating complex diseases or altering traits in humans that are influenced by more than one gene [7]. In April 2015, Chinese scientists published their work on CRISPR/Cas9 genome editing 1 2

Transcription activator-like effector nuclease Zinc-finger nucleases

Nanoethics

in human embryos [1]. They used the technique to repair a mutated ß-globin gene which causes β-thalassaemia, an inherited blood disorder. As the embryos they used were tripronuclear – which meant it was clear from the beginning of the experiment that the zygotes would not be able to develop normally – no clear-cut conclusions may be drawn from these experiments with regard to the feasibility and safety of genome editing in humans [1]. The paper stirred up a new debate about the ethical implications of germline editing and prompted leading scientists to call for a moratorium, which recalls to mind the situation in the early days of genetic engineering in the 1970s [8, 9]. A statement by the scientist and genome editing advocate George Church illustrates the perplexity felt even by expert scientists when considering the prospect of CRISPR/Cas9 genome editing in humans: BWhat is the scenario that we’re actually worried about? That it won’t work well enough? Or that it will work too well?^ [10]. On the one hand, there are concerns about the safety of a therapy that alters the plan of each body cell in a human being. Such a therapy might prove irreversible and, in the event of unforeseen and harmful side effects, could pose a threat even to future generations. On the other hand, if CRISPR/Cas9 could be applied safely in the future, issues of injustice and accessibility might arise due to the likely high price of germline therapy, increasing the relevance of general ethical objections about enhancement and fear of eugenics. In my opinion, both the concerns raised by Church are relevant, yet the complexity of the topic requires a nuanced consideration of what exactly might not work well enough, and what exactly it is that we do not want to work out, in order to establish what action should be taken to prevent undesired societal effects, ethical issues and harm to health. An assessment of the technological state of the art of CRISPR/Cas9 germline editing and its ethical implications in the light of today’s social, economic and legal context will reveal that some of the concerns already raised in the 1980s [11] should be discarded as too visionary and speculative, whereas other issues have emerged that should be considered more seriously. The aim of this article is first to take a brief look at the arguments of different interest groups, each with their own background and interests, to see which distinctions, frames and focus they use, and to point out possible shortcomings of each of these perspectives. Based on the state of the art and ELSA (ethical, legal and social

aspects) of CRISPR/Cas9 germline editing, a scenario illustrating the future use of CRISPR/Cas9 in humans will be developed and relevant ethical questions and regulatory challenges identified. A consequentialistic line of argumentation will be used that stresses the rarely considered socioeconomic and practical challenges of genome editing for medical purposes. Ways to open up the narrow discussion bring in new perspectives will be proposed by illustrating examples.

Distinctions, frames and comparators used by participants in the debate Synthetic biology can be compared to either biotechnology, information technology or nanotechnology, with different outcomes for the judgment of the new technology [12]. Depending on the chosen comparator, different discussion frames will predominate, e.g. ethics, risks or potential benefits [12]. The comparison is not so much about the details of the methods or aims, but about framing and emphasizing the benefits for major problems (nanotechnology), personal life (IT) or risks (biotechnology). For example, one might claim that synthetic biology is an extended or more sophisticated form of biotechnology, and as such is associated with increased risks due to the use of more radically modified organisms. Synthetic biology can also be compared with nanotechnology, which was primarily discussed with regard to potential benefits in the past. [12] One might then suggest that synthetic biology is a technology that will have even more impact on our lives than nanotechnology by offering solutions and benefits to major problems of humanity like disease management and food scarcity. Considering the dominant frames and comparators used by different groups or stakeholders can also help us understand controversies in the CRISPR/Cas9 debate. The following presentation of weaknesses or biases of different views should in no way be understood as a lack of appreciation of those views. The combination of different views is crucial for fruitful discussion, and no moral viewpoint can be inherently wrong. However, it is important to identify differences in values or interests between discussion-leaders and the rest of the society in order to prevent some relevant arguments and perspectives from being neglected and others from being overrepresented.

Nanoethics

The following observations about the discussion of CRISPR/Cas9 and its ethical implications are based on a small number of articles from leading, mostly German, newspapers, viewpoints expressed by some leading scientists in interviews [8], and a non-extensive body of bioethics literature on germline editing and synthetic biology which implicitly includes human genetic engineering. Ethics councils are currently still working on ethical and governance guidance [13], while articles referring to ethical implications of CRISPR/Cas9 are still limited to blog contributions or short comments [10, 14–18] and opinion articles [19–21]. More comprehensive articles discussing the ethical implications of novel genome editing technologies are rare and either consider a broad range of applications [22] or focus on legal and regulatory issues [23, 24] of human germline editing. Nevertheless, certain tendencies with regard to the framing of arguments, distinctions drawn between different applications or their purposes, and comparisons with other technologies can be observed. The intention of this part of the article is to make it clear that the arguments of different discussion participants might be shaped considerably if they had different values, interests and knowledge. Mass media: New therapies vs. risk When a Chinese research paper reporting the genetic modification of human embryos [1] was published in April 2015, the media coverage included not only positive visions of curing diseases (Spiegel [25]) but also negative scenarios of a world of ‘designer babies’ (Focus [26]) and references to hubris (Tagesspiegel [27]). It was felt for the most part that these experiments had broken with an ethical taboo, as the gene pool or identity of humanity might be threatened by the heritability of the treatment and its unpredictable damaging side effects (SZ [28]). However, a future application for the eradication of genetic diseases, a kind of Bsteering evolution^ (Zeit [29]), was seen as challenging, but quite likely (National Geographic [30]). Countering calls for caution, the opinion was put forward that progress should not be blocked because CRISPR/Cas9 could give people healthier, longer and better-quality lives (Economist [31]).

Scientists: Basic research and somatic genome editing vs. germline editing Some scientists draw a distinction between the clinical application of germline editing on the one hand and somatic gene therapy or basic research with CRISPR/ Cas9 on human embryos on the other. At the time no clinical application of germline editing that might offer any additional benefit over alternative existing methods was conceivable [9], yet other uses of CRISPR/Cas9, such as somatic gene therapy, should not be categorically rejected or over-hastily restricted [9, 32]. Basic research, though also related to ethical issues like embryo wastage, egg cell donation or laboratory animal use, promised important findings relevant to the treatment of human diseases, and was also justified by the freedom of research [8]. Obviously scientists might be concerned about strict regulations governing basic research and somatic gene therapy - such as the existing embryo protection laws that forbid the use of embryos for any research purposes in some countries, including Germany [23] – as these could block research, progress and innovation. To prevent this heated debate leading to any such outcome, they use the comparator gene technology and the Asilomar conference [33], pleading for a moratorium [9, 32] and thereby showing their willingness to limit research activities to accepted areas and to engage in open discussion. They vote for a more specific regulation rather than a general ban on research into any specific technique or into human therapeutic applications in general. However, the distinctions made between somatic and germline editing, as well as between basic research and clinical application, may still be too approximate. A nuanced view must be taken of the uses of CRISPR/Cas9 for different therapies and different basic research purposes and of their respective benefits, risks and ethical issues. Although it is legitimate for a scientist to attribute considerable importance to the possibilities offered by research, this inevitably leads to less weight being attached to the ethical and economic implications for clinical practice that are revealed only if a more differentiated view is taken. Theoretical arguments, questions and recommendations in bioethics literature Bioethicists display a wide range of opinions about the desirability and feasibility of germline editing and pose questions and formulate tasks for bioethics that will be

Nanoethics

further pursued in this article. Miller believes that the process-based guidelines which resulted from Asilomar were not a success but cause science to struggle, and thinks that CRISPR/Cas9 could conceivably eradicate sickle-cell disease, a Bmonstrous genetic disease^, meaning that any ban on human germline therapy would be unethical [18]. Opposing this view, Krishan et al. focus on the issue of eugenics, stating that the Bneoeugenic era where diseases can be eradicated with a possibility that individuals will be cured or designed even before birth (…) certainly calls for another Asilomar Conference^ [19]. Kaebnick is less confident about the feasibility of therapeutic applications of germline editing. The great challenge in all potential applications would be to make the correct genetic changes, yet CRISPR/Cas9 as such constitutes merely a technical revolution that helps to make the genetic changes correctly [34]. Questions were raised about what, if anything, was exceptional about research into CRISPR/Cas9; in order to find answers, it was supposed that it will be necessary to underscore normative aspects, separate science from fiction [82] and learn from previous debates [21]. Publications on the ethics of synthetic biology predating the wide application of CRISPR/Cas9 discuss deontological arguments [35], the injustice argument [36] and utilitarian reasoning [37, 38]. According to them, there can be no rejection of synthetic biology applications based on fundamental moral reasons. They also dismiss as inappropriate the precautionary principle, which may be invoked when a technology may have a dangerous effect, identified by a scientific and objective evaluation, if this evaluation does not allow the risk to be determined with sufficient certainty (European Commission definition). Although these publications are only three years old, the ethical discussion may need to be updated in order for adequate consideration to be given to new technical developments in genome editing, and to determine whether the assumptions made in these arguments are rather reinforced or invalidated by further developments in CRISPR/Cas9 technology and its initial use in human embryos. For example, it was argued that the cheapness of a technology invalidates the argument of just allocation of resources, at least in the long run [38]. CRISPR may indeed change assumptions regarding costs and also regarding safety, and a parallel gain in knowledge through genomics may open up more applications for the technology in humans, both for health care and enhancement purposes. CRISPR technology and its first

use in human embryos may actually be so entirely new that scientists, media and bioethicists have to call it a breakthrough on the one hand yet assume on the other that the experiments mean crossing an ethical line. The question I wish to pursue is whether the benefits and/or risks of CRISPR/Cas9 germline editing are really so much greater than those of existing technologies in reproduction medicine, and what implications this has for a consequentialistic account and with regard to the precautionary principle. Generally speaking, the observations from the literature and mass media emphasize the moral gap between therapy and enhancement while concentrating on the risks and dangers, though also on the potential therapies for incurable diseases. Most of the articles do not contain any more detailed report of the Chinese results nor any more nuanced view of ethical aspects. The emphasis is clearly on attention-grabbing emotional content rather than detailed information. CRISPR/Cas9 genome editing is compared by many scientists with genetic engineering and embryo research in an attempt to highlight the risks entailed in limiting research possibilities and to demonstrate moral responsibility by referring to the openness of the early genetic engineering debate. The preferred frames are risks and ethics, though benefits are also stressed. Bioethicists not only focus on the risks to individuals or humankind; they also discuss ethical deontological arguments like autonomy [35, 39]. These discussions often fall short of providing a detailed analysis of different applications and their technical feasibility, and instead discuss the ethics of germline editing as such. Although economic aspects are mentioned in the context of just access [38], somewhat speculative assumptions are made about scientific, technical and practical feasibility and acceptance in society. The theoretical viewpoint also prevents the consideration of ethical challenges arising from clinical practice. In summary, the most prominent voices in the current debate do not mention the economic and practical challenges in the clinic. These perspectives will be discussed below, and could gain relevance in the public debate when different comparators are used. Germline therapy could be compared to pre-implantation diagnostics (PID), which poses regulatory challenges for authorities regarding the definition of an indication catalogue and decision-making dilemmas for parents. Somatic genome editing could be compared to antibody therapy, an expensive health technology which offered benefits to a

Nanoethics

limited number of patients at the time. Until quite recently, antibody therapy was discussed primarily with regard to its benefits and as a more targeted and effective therapy than alternative chemical agents. Now, however, the socio-economic considerations of this expensive technology are being increasingly taken into account in health technology assessment. Gene therapy, whether it targets the germline or somatic cells, could be seen as the next generation of personalized medicine. Rather than focusing again on the benefit frame, however, the economic frame could be considered from the beginning. Historical accounts about the development of IVF may suggest that it is pointless to impose strict regulations or bans on emerging medical technologies, and may help shift the debate away from the simple question of whether to ban or allow them and towards more pragmatic solutions.

How to tackle the complexity of the debate As regards other techniques which are linked to assisted human reproduction or affect heritable traits, namely pre-implantation diagnostics (PID) and mitochondrial transfer, the list of arguments for and against human germline editing with CRISPR/Cas9 may be quite long, but is not fundamentally new. Rather it seems that ethical concerns are repeated every time a new technology emerges, disregarding the fact that the older technology has meanwhile come to be widely accepted. This observation implies that little is learned from previous discussions, that certain arguments are simply ignored by regulatory authorities, clinicians and patients, or that moral feelings and judgments are relatively flexible and change over time. This may well be the case given that arguments in controversial debates are by definition often dependent on unreliable estimates and normative assumptions. How is an appropriate level of safety to be defined? Is a regulatory distinction between therapeutic and enhancement purposes desirable? Is there any justification to abandon human germline therapy altogether because of general moral concerns regarding autonomy, naturalness or identity? These questions can only be answered by engaging in a large amount of speculation and subjective values. Ethical dilemmas will inevitably arise: between the freedom of science and moral standards, and between the duty to cure disease and the obligation to prevent harm to scientific research subjects.

In the present debate on germline editing, however, there is much to be gained by analyzing the arguments of different stakeholders with respect to their contextspecific or interest-driven assumptions and weightings, as has been briefly outlined above. Bioethics should attempt to add missing interpretative frames and comparators so as to be able to take into account all perspectives and at least pose, though obviously not conclusively answer, all relevant ethical questions. A highly pragmatic ethical framework will be defended in this article, although human rights (autonomy, freedom, dignity) naturally have to be discussed and ensured when new medical technologies are used. Deontological arguments will not be discussed here, however. Instead, the focus will be on costs versus benefits, alternatives, and other ends that the resources could be used for, as I believe that the socioeconomic and implementation issues have not been discussed widely enough as yet. Another important prerequisite for a meaningful debate is that facts and fiction be separated [82], i.e. that arguments be based on realistic assumptions about the technological development and its potential for applications. This procedure will be outlined below.

Prospects for different therapeutic applications of CRISPR/Cas9 in humans Basically, there are three different aspects that need to be considered in order to predict the feasibility of clinical applications of CRISPR/Cas9 genome editing: 1) the technical feasibility of modifying the genome, 2) knowledge about disease-causing sequences in the genome, and 3) the capability to diagnose genetic diseases. Prerequisite 1: Technical feasibility The technical aspects of modifying the genome are efficiency, i.e. the rate of success (the proportion of cells in an embryo that have been modified), and precision, i.e. ensuring that only the intended sequence is modified in the desired way. Currently, the genetic modification of an embryo in vivo, i.e. in the womb of the mother, appears to pose an even greater challenge - due to the additional difficulty involved in delivering the CRISPR/ Cas9 system to the cells inside the body - than an ex vivo modification and implantation of an in vitro fertilized (IVF) embryo. Thus no further consideration will

Nanoethics

be given here to in vivo embryo editing. A female is capable of producing a limited number of egg cells in one fertility cycle, and IVF has a success rate, measured as the life birth rate, of only 20 % per cycle [40]. Modification efficiency must thus be very high in order not to further decrease the overall success rate to a point at which the procedure, which is physically and psychologically stressful, becomes unbearable for mothers. Besides efficiency, it is important to prevent what is known as mosaicism - a phenomenon in which not all body cells are successfully modified and which could therefore limit therapeutic effect. Furthermore, high levels of precision are necessary to exclude modifications of non-intended sequences, as these may cause harmful side effects. These requirements have not been met so far. Monkey zygotes have been edited with an efficiency of around 20-30 % with CRISPR/Cas9, though this has only been achieved for the insertion/deletion genetic modification type, which is therapeutically less relevant than correction. The study was also small, comprising only two successfully modified twins which were born healthy [91]. Gene correction in zygotes was achieved in mice with a low efficiency rate of below 5 % [92]. When two or three modifications are performed at different genomic sites, efficiency rates decrease further to about one half of the efficiency of a single locus modification [23]. The only published study involving human embryo editing reported an efficiency of 15 % for single gene correction [1]. Off-target frequencies and mosaicism have been found [1]; these are side effects which are crucial to know in detail with respect to clinical applications, but which are difficult to assess [5]. There are huge differences in efficiency depending on the animal species, genome editing technique, delivery method of the genome editing system, and type of sequence modification [23], which means that it is still difficult at the current time to make any general predictions about future developments and successful strategies. Although somatic cell therapy using CRISPR/Cas9 is considered ethically less problematic as it is not passed on to future generations via the germline, it still poses technical challenges and also entails safety issues [41]. Technical requirements for the modification of the genome of body cells are not only the efficient and precise correction of a genetic mutation, but also the successful delivery of the CRISPR/Cas9 system to the target cells, which is still difficult due to its large size [42].

Prerequisite 2: Knowledge of genetic causes of diseases and determinants of body traits The success of any genetic therapy also depends on whether the exact genetic cause of the disease to be treated is known. Such knowledge differs hugely depending on whether it is a question of enzyme deficiencies on the one hand or diseases of complex etiology such as diabetes, cancer or psychiatric illnesses on the other. Both classes of disease may have genetic causes, yet the number of relevant genes in the case of complex diseases is often high and mutations with a major impact are rare, as are patients suffering from a complex disease caused by a single gene [43]. Despite the explosion of genomic sequence data, science is still far from understanding the genetics of complex traits such as intelligence or even body height. [44] As far as enhancement is concerned, however, single genes offering benefits in terms of bodily functions or appearance, such as athletic endurance or eye color [44], do indeed exist. Prerequisite 3: Diagnosis of genetic disease The application of germline editing is currently only considered in a very limited number of special cases [23]. Couples concerned would have severe genetic illnesses themselves, and would know from their disease symptoms, genetic screening or family history that they were at risk of developing diseases later in life. The detection and correction of homozygous genetic predispositions to disease would pose additional challenges as such predispositions are not necessarily evident from one’s family history. Nonetheless, PID is no alternative as all this couple’s embryos would be affected. For germline editing to become superior to PID in other cases, the CRISPR/Cas9 technology would have to make huge improvements, e.g. would have to allow multiple modifications. To be applicable, the diagnosis of genetic mutations in embryos would need to be as safe and reliable as modification. Another application in principle would be to protect an embryo from de novo mutations that naturally occur in egg and sperm cells or that originate during the embryo’s development. Embryos would have to be screened as a precautionary measure even where no severe mutations were generally expected on account of family history. Again, extremely high standards would be needed to assure the safety and accuracy of genetic screening. Even with state-of-the-art

Nanoethics

techniques, however, whole genome screening is still unreliable [45, 46] and costly. Recalling the cited statement by G. Church, the technological advancement of modification and genetic screening would reduce certain problems such as the safety issue, but would generate others, such as injustice due to high prices or by lowering thresholds for applications considered unethical. According to the slippery slope argument, it might be tempting to at least correct the most severe mutation in an embryo that had already been screened, e.g. a mutation that causes an undesirable physical trait, even if there were no mutation causing severe disease.

Risk-benefit analysis of CRISPR/Cas9-based germline therapy To develop a valid consequentialistic argument based on facts, the potential harms and benefits of germline therapy, as well as its costs, need to be considered. Given the state of the art as outlined above, the most realistic use of germline editing would be to prevent a genetic disease that is already present in one or both parents and that would definitely be passed on to the child without medical intervention. In the case of autosomal recessive diseases (e.g. cystic fibrosis, phenylketonuria) where both parents are homozygous, or autosomal dominant diseases (e.g. Huntington’s disease, familial adenomatous polyposis) where at least one parent is homozygous, germline editing would be the only way to have a healthy child that is genetically one’s own [23]. This is not a problem that can be solved by PID and the negative selection of embryos with the disease allele(s) [23, 47]. Since alternatives exist even in these cases – such as choosing not to have a child, adoption and egg cell or sperm donation – the critical question is whether the benefit of having a child that is genetically one’s own is sufficiently high to justify the risk of potential harm, to the child itself and to future generations, that may result from modifying the genome. In a study on the attitude of reproduction medicine experts towards mitochondrial transfer, another germline modifying technique, many of the interviewees Bdid not believe that to have a genetically related child, although very important to most people, was a right or a sufficient reason to develop and use experimental techniques.^ [47] Others

pointed out that the desire to reproduce and have genetically related offspring is hardwired in us by evolution, and that this can only be partly overcome by societal measures such as easier access to adoption and egg cell donation [47]. In the case of medical indications where PID is an alternative to germline modification and entails the same benefit, i.e. a healthy child that is genetically one’s own, one may compare the risks of different treatments. It is also important to keep in mind that parents may risk having a diseased child when this is the only possible means of having a child which is genetically their own. The harm that this decision may entail for the child may – depending on the severity of the disease – still be less than the potential harm that germline therapy poses to the child. The benefits that germline editing might offer are just as difficult to measure objectively as it is to quantify such risks. Nonetheless, attempts have been made to quantify the benefits and evaluate costeffectiveness of medical treatments using the willingness-to-pay measure, which is based on the amount of money that people claim they would spend on treatment, or according to the QALYs (quality-adjusted life years), the years of life gained, adjusted for health-related quality of life [48]. Safety is also an issue when it comes to somatic gene therapy using the CRISPR/Cas9 system safety, though the risk of potential harm is considered to be generally lower and more predictable, and clinical guidelines may be applied that are already being used for gene therapies based on other modification techniques. [49–51] According to economic theory [52], a distinction should be drawn between risk, which is calculable, and uncertainty, where no probabilities can be stated, or which is an unknown. It may be argued that germline safety issues are primarily about uncertainty, as there is no knowledge of the likely incidence of risks due to the lack of previous experience; what is more, there may be entirely unforeseeable side effects. For most people this is enough to justify the precautionary principle with regard to germline therapy, though there are also contrary opinions. I will take Smith’s [38] argumentation as a starting point to show that some debatable assumptions are used when arguing against the precautionary principle and that a technique like germline editing should not be treated in too undifferentiated a fashion regarding applications, their safety and costs, if the aim is to identify relevant ethical implications.

Nanoethics

Consequentialistic argument based on benefit, risk and cost Disregarding risk and uncertainty, the benefit should be measured and compared with existing treatments and their costs for socio-economic reasons if implementation in the real health care system is the goal. Costbenefit analysis is a method used in health technology assessment which can be applied on different levels of the health care system, from individual hospitals to the health care system as a whole [53]. To date, the costs of new technologies have rarely been discussed openly when it comes to ethically contested therapies such as germline editing, and indeed somatic gene therapy. However, the socio-economic dimension of new therapies should not be neglected as it has ethical implications for just access to and allocation of resources [38, 54] and because it has practical relevance when estimating the likelihood of a Bproof of concept^ technique being translated to the clinic. The ethical concern about just access to new technologies is sometimes refuted by arguing that unjust access is a characteristic of every new technology at first, and that it will vanish in the long run as the technology becomes cheaper and eventually benefits society as a whole [38]. Two erroneous assumptions are made when this argument is put forward in the case of CRISPR/ Cas9 germline and somatic therapy, however. First, there is no substantial price decline in biologically derived therapeutics, as there are no generic drugs in this sector on account of the differences in production standards as compared with simple chemical substances, which means that companies other than that which initially developed the therapeutic in question are unable simply to mimic the production process, or at least not at a much lower cost. Second, Smith’s argumentation is based on the assumption that everyone will eventually benefit from gene therapy through enhancement, which demands both a broader public acceptance of this goal and its feasibility, which is mostly limited by knowledge rather than by technology alone [44]. It is likely that germline therapy, if ever made available, will considerably strain the public healthcare budget, as can already be seen with Glybera, the first approved genetic therapy in Europe, which is priced at $1 million [55]. Such therapies may thus become a private luxury; although they cannot be morally condemned as such, their development probably should not be publicly funded from the viewpoint of the just allocation of resources. A detailed

cost-benefit analysis of gene therapies may be needed when the number of possible research trajectories further increases and the investment sums needed to realize them rise with them. The simplest way of conducting a cost-benefit analysis is to directly compare treatments with the same curative effect, e.g. the genetic disease factor IX deficiency/haemophilia B can be treated by means of gene therapy or protein infusion [3]. Having advantages in terms of efficiency and production, CRISPR/Cas9based therapies may be cheaper than genetic therapies based on other genome modifying technologies and protein therapy. Besides this single case-centred view – comparing different technologies that offers the same patients the same benefit – a broader socio-economic view can also be adopted. In this case, the important questions involve asking how many people will benefit by how much, and how high the necessary research, development and application costs are. This ratio must then be compared to the benefit-cost ratio of other therapies for the same diseases, as well as for entirely different diseases. Regarding CRISPR/Cas9 germline therapy, Balleviating pain of infertile couples should always be balanced (…) against other needs in society that may have a more convincing claim on the healthcare budget.^, as experts have stated with regard to mitochondrial transfer [47]. It can be estimated that the number of people who could benefit from CRISPR/Cas9 germline editing and who have no other means of having a healthy child that is genetically their own is negligibly low [44]. The costs of germline editing could be roughly estimated on the basis of current technologies such as IVF, PID and mitochondrial transfer, and should also include safety trials, follow-up studies and diagnosis. Two critical questions should be posed when it comes to a comparison with other diseases and treatments: First, whether money is spent on the Bright^ diseases, which of course does not imply that orphan diseases (from which only small minorities suffer) have no rights whatsoever to receive financial aid. It is not necessarily morally better to treat a large number of mildly ill patients than one fatally ill patient. The point is that there may be diseases that are equally severe, but which affect much larger numbers of patients who could be treated with a higher success rate and less money than patients with monogenetic diseases who could potentially be treated by germline or somatic genome editing. The development of new antibiotics or the monitoring of

Nanoethics

hygiene standards in hospitals could serve as one such example, considering that this could save 25,000 lives and 1.5 billion euros [56]. The second question is whether a specific disease is tackled efficiently. Overall, a marked trend towards allocating resources to expensive therapeutics generated by high-tech methods can be seen in the pharmaceutical sector, e.g. the proportion of antibody therapeutics on the market for new cancer drugs has increased and the effectiveness of the resulting therapeutics has been rather small measured against the investments [57]. Although significant improvements have been achieved, breakthroughs in terms of patient benefit have also failed to materialize so far despite two decades of research and trials of gene therapy [58]. Of course, this fact does not invalidate the right of freedom of research, nor does it mean that the aim of developing gene therapeutic applications of CRISPR/Cas9 is not justified. However, it is necessary to think carefully about which projects deserve how much funding if they explicitly aim to maximize benefits for a country’s society as a whole. The most realistic application for CRISPR/Cas9 somatic therapy is the Btreatment of monogenic, highly penetrant diseases, such as severe combined immunodeficiency (SCID), haemophilia and certain enzyme deficiencies, owing to their well-defined genetics and often lack of safe, effective alternative treatments^ [3]. This application promises high benefit but will help only a small number of patients as compared to those suffering chronic and multi-factorial diseases such as cancer, where effective treatments could benefit millions of people. This makes cancer an attractive target for pharmaceutical companies and makes funding of research easy to promote. However, an effective cancer therapy is often much more difficult to develop than many therapeutics to combat monogenetic diseases. According to the clinicaltrials.gov database, a large proportion of ongoing gene therapy trials targets cancer or other complex diseases, yet the first approved drug was designed to treat a monogenetic disease [59]. A large proportion of clinical and experimental gene therapies is for a cancer indication, which should not give the impression that this is the most promising application, as chronic diseases are mostly multifactorial and are also affected by multiple genes that are not easy to identify and correct due to their number and interindividual variability. Potentially promising applications of CRISPR/Cas9 should always be compared to existing

alternatives to this expensive high-tech solution, and should be done separately for every specific disease. One possible conclusion from the benefit, cost and risk analysis, as unethical as it may appear at first glance, might be that certain kinds of genetic therapies are simply too costly to be applied from a just distribution viewpoint. Smith’s consequentialistic argumentation that was taken as an example is based on contentious assumptions regarding the acceptance, scientific knowledge and technical and socioeconomic feasibility of germline editing. CRISPR has been praised for its cheapness and ease of use, but that is only true of most basic research purposes and is only one enabling factor for gene therapy, which depends on many other medical, technical and economic factors. Future investments in genomic research and the health care system should be made in order to create and deliver benefits for society as a whole [60].

CRISPR/Cas9 in basic research According to the argumentation that has been put forward in this article thus far, application-oriented research on human germline editing should not be publicly funded due to socio-economic considerations and safety issues. This appears to be in line with some leading scientists, who propose a moratorium on human germline editing. However, the ultimate aim of this call for an open discussion is to prevent restrictions on basic research into CRISPR/Cas9, including experiments on human embryos [9, 32], due to its substantial potential for improving understanding of human development and genetics, and for finding substances to tackle complex diseases [2]. Despite calls for a moratorium also including basic research on human embryos with CRISPR/Cas9, however, such experiments are already being planned in the UK [61]. Monkey germline editing is not subject to any particular restrictions and may become more widespread in the future given the technical ease of using CRISPR/Cas9 in higher mammals [62, 63]. Although not intended to develop the technique for human germline modification, research of this kind will inevitably help improve the efficiency and precision of CRISPR/Cas9 and bring science a big step closer to achieving technically feasible germline editing. It may also reveal new potential targets for genetic correction. Viewed positively, this makes the technique safer and provides knowledge for novel therapies. However, the

Nanoethics

view that basic research will have no practical implications for human germline modification may simply be too naïve and illusionary. If the taboo surrounding human germline modification is to be taken seriously, certain directions in basic research should be carefully considered and probably restricted in advance before Bdangerous knowledge^ [20] is produced that will inevitably lead to it being applied in a clinical context at some point in the near future. However, it is also questionable whether this is possible at all and whether it would not be better to take more practical measures to make clinical implementation as safe and beneficial as possible for patients.

Regulatory challenges – total ban on germline editing based on the safety argument vs. indication catalogue There is broad consensus that the current technological state of the art does not allow CRISPR/Cas9 to be applied in the human germline for reasons of safety, and leading scientists believe that any further experiments in this direction should be stopped until conclusions regarding fundamental ethical objections are reached in an open debate [8, 9, 32]. But how is germline editing and associated research actually regulated by law at the present time, and which changes might be needed to close regulatory gaps or resolve grey areas when technical developments emerge in the future? While existing regulation differs internationally, some countries allow embryos to be created, whereas others only permit research involving surplus IVF embryos [24]. Some scientists argue that embryo modification with CRISPR/Cas9 does not differ fundamentally from any other kind of research on embryos, provided that they are discarded after use and not implanted [61]. Although research funding of germline editing was recently prohibited in the US, privately funded research is subject to little oversight [64]. Many European countries ban human germline modification [23]; in Germany for example this is enshrined in paragraph five of the embryo protection law [65]. The United States and China do not have any laws to regulate this therapy, but any attempts to establish such a pregnancy would require the approval of the U.S. Food and Drug Administration, and any clinical use is prohibited by the Chinese Ministry of Health guidelines [10]. Prenatal therapy, i.e. in vivo germline

modification, is less regulated and not covered for example by current German law, but is also further away from reaching technical feasibility [65]. While concerns are expressed in the mass media [28] that it may be unfeasible to control CRISPR/Cas9 germline editing given how easy and cheap the system is to use, these are not justified because high-tech equipment and expertise is needed to handle human embryos [8]. Although existing regulation appears to be sufficient, it is important to keep in mind that ongoing and future developments of genome editing techniques, and possibly also clinical applications in less strictly regulated countries, may take place sooner or later and, if successful, may be judged sufficient to invalidate the current safety argument in favor of a total ban of germline editing [65, 66]. Such a course of events would not be unprecedented in the history of IVF, where private funding led to the birth of the first in vitro fertilized child in the UK, and the artificial reproduction technique became rapidly accepted by funding authorities and in practice soon afterwards [67]. Private companies and hospitals might then begin to specialize in this kind of therapy, as is already the case with PID. Widespread use of germline editing is not very likely due to its high costs and the probable refusal of health insurance companies to pay for it unless it can be proved to guarantee savings in the future, e.g. lower spending on alternative therapies for cancer patients or non-fatal monogenetic disease patients. Nonetheless, private health insurers with higher budgets might offer this therapy, or services might be offered to private clients by specialized companies. Considering such a scenario for germline editing, a total ban based on arguments such as dignity or autonomy would have to stand up against the moral obligation to promote medical intervention in the case of diseases that lead to great suffering of patients. Rather than a complete ban, restricting germline modification to specific indications would probably be the most practical solution to ensure that both ethical requests are met.

Possible content of an indication catalogue for germline editing In view of the current technological state of the art, the only therapeutic germline editing application without alternatives would be to allow parents with a specific

Nanoethics

constellation of genetic predispositions to monogenetic diseases to have a healthy child that is genetically their own [23]. This benefit could be considered sufficient to justify some non-excludable risk of germline modification, as is already accepted in IVF, PID and mitochondrial transfer in the UK. Depending on an assessment of the potential harm caused by germline editing as compared to PID, a wider or narrower indication catalogue could be defined. PID involves taking a cell from the embryo; a mechanical procedure that could theoretically harm the embryo. In Germany, this is not permitted for any indications other than severe monogenetic diseases that will emerge with a high degree of certainty before adulthood. Ethics commissions decide on a case-bycase basis whether the genetic predisposition of the parents justifies the selection of an embryo to prevent transmission of the disease-causing mutations. Regular reports and discussions of clinical experience of PID in recent years are designed to prevent the range of indications from being widened to include anything apart from very severe genetic diseases. It is possible that the indications could be widened given that genetic diseases are rare and knowledge about them is still growing, yet a shift towards less severe diseases must be prevented according to the state ministry for health [68]. Currently, due to potential off-target effects, mosaicism and inefficiency, germline edited embryos would need to be screened in addition to being modified, which would give rise to an additional risk factor. Consequently, the indication catalogue should be even narrower than for PID. In principle, however, huge developments could make genome editing as safe as PID, and eliminate the safety disadvantage which currently makes PID the better alternative for most indications. Furthermore, germline editing opens up new possibilities due to the introduction of variants that do not even exist in the parents; this produces new potential benefits that could outweigh any potential harm. Based on these assumptions, more deliberate regulation could allow modifications to provide protection against monogenetically caused forms of cancer or Alzheimer. When screening becomes cheaper and the interpretation of genomic data more automated, and as the association of genomic variants with disease proceeds, it may even prove tempting to also allow genetic variants to be corrected that are thought to have a high probability of having a severe impact on health. Such parental predispositions do not have to be screened for in the embryo itself but can be detected by screening egg cells, sperm cells or even

body cells. That would make application easier as parents who are generally open to germline correction would not have to expose their future child to risk by screening on spec.

Germline therapy – a challenge for clinical trials Is it feasible and ethically acceptable to adapt guidelines that regulate conventional clinical trials to include germline editing? Or is it impossible to reach an acceptable safety level given the exceptional characteristics of this kind of therapy? Germline editing affects all body cells, including sperm or egg cells, and will be passed on to future generations. This is often put forward as a catch-all argument against any intended modification of the human germline, as the risks will not be known until the first experiment on humans has been done and its consequences will be irreversible for the child and maybe even its descendants. In contrast to other experimental treatments that are justified by the argument that the harmful side effects are most probably smaller than the harm posed by an untreated existing disease, there is at least one alternative to germline editing, namely the decision not to have a child that is genetically one’s own in the first place. This kind of problematic situation is not entirely new. Although established in clinical practice, PID could also lead to heritable changes by epigenetics [69], and IVF/PID children still have not reached sufficient age for one to be absolutely sure that the procedure has no adverse effects in older age [70]. Generally speaking, every new health technology or therapeutic substance has certain unpredictable risks, that is to say a degree of uncertainty about potentially harmful side effects always remains to some extent. This fact can be used as an argument both for and against any new technology. There are historical cases in which concerns raised at the time of introduction seem ridiculous today and others in which greater caution would have been a good decision in hindsight. And there are also examples where attitudes changed from precautionary to risk-taking in view of the demonstrated benefits, as in the case of IVF development which was denied state funding until initial privately funded medical successes led to rapid acceptance by regulatory bodies, clinicians and patients [67]. Regarding clinical applications of CRISPR/Cas9, the fear of underestimated or overlooked risks could be fueled not only by media reports which warn of the first

Nanoethics

‘designer babies’, ‘Frankenstein’ experiments or threats to the human gene pool, but also by well-known cases in which therapeutics were launched prematurely because regulatory procedures failed and harmful side effects became evident only following market release, as for example was the case with thalidomide [71]. The high investment costs of clinical trials may lead to economic pressure on pharmaceutical companies and rather discourage a more precautionary approach than is dictated by law, which is why it is the job of regulatory bodies to ensure that patients are protected. Clinical trials of chemical substances, biomolecules or even somatic gene therapy using editing techniques are designed and able to ensure the health of the test subjects in principle because therapeutics will degrade sooner or later and can be administered in controlled doses [49]. Germline modification is an irreversible and all-or-nothing procedure. Apart from the issue of the impossibility of informed consent, entirely new clinical guidelines would need to be designed, for example with regard to the number and duration of experiments on monkeys and of the clinical parameters to be tested so as to replace human trials to the greatest possible extent. However, since even those species of monkey that are most similar to man might still behave differently, an additional safety measure which might be considered could involve limiting the number of people treated in the first trial and comprehensively monitoring them for some years before allowing the treatment to be applied to a larger group of patients. By way of highlighting that this kind of procedure is by no means self-evident, mitochondrial transfer – which can also be classified as germline modifying therapy – was previously allowed in the UK following a very limited number of trials on monkeys [72], a decision that could be criticized as too hasty. Trials of somatic gene therapy with CRISPR/Cas9 could already become a reality in just a few years’ time. On the one hand, guidelines have already been adapted for other types of gene therapies which affect somatic cells, and should be sufficient for CRISPR/Cas9 in principle. On the other hand, the design of these guidelines may need to be improved in order not only to allow affordable therapies to be developed but also to protect patients from adverse side effects and non-effective therapies. Due to the novelty of gene therapeutics in clinical practice and a lack of experience of risks, guidelines require many additional tests which are not stipulated

for conventional therapeutics, such as an analysis of DNA or protein expression, which could be altered in unexpected ways by gene therapy [73]. One challenge will be not to exaggerate the safety and production standards so that gene therapies can be affordable while at the same time protecting patients from adverse side effects. Furthermore, effectiveness has to be ensured so as to limit unnecessary health care expenditure and protect patients from ineffective therapies that delay cure. One challenge here is that gene therapies often represent experimental therapies that are granted a special permit for use and do not fall under the law which regulates other therapies. Cancer therapeutics are increasingly used off-label without clinical efficacy trials for indications other than those for which they were initially tested [74]. Though not necessarily posing a health risk, this is an example of how clinical trials and medical practice can fail to provide patients with effective therapy yet at the same time represent an additional burden on health care economics.

Challenges for clinical practice – consultation and remaining decision conflicts Returning to the issue of legalization and an indication catalogue for certain therapeutic modifications, it is likely that regulatory authorities will nevertheless always leave it to the parents to take the final decision for or against a therapy in order to protect their autonomy. Diagnosis might also depend on parents’ decisions given that genetic screening, depending on the disease in question, may prove expensive and may not be paid for by most health insurers. This is a considerable challenge for consulting clinicians and future parents due to the complexity of the options and the potential benefits and risks of future genetic medicine. Such situations already exist with PID but may become relevant for a larger number of people if it becomes possible to modify genetic variants as well as select existing ones. Technological possibilities can force parents to decide and impose huge responsibilities on them. The right not to know and to leave difficult decisions to nature may become unaccepted in society. Parents who decide to accept the potential consequences of a disease which can diminish quality and capacity of life rather than face the risks of germline modification or the high costs of such therapy may be challenged to justify their decision.

Nanoethics

Concerning the more visionary correction of de novo mutations, diagnosis as such would be a risky procedure that would lower the chance of a successful live birth of a genetically tested embryo. In other words, a decision which could compromise the future child’s health has to be taken even before therapy is actually carried out and without knowing whether therapy will be needed at all. In cases where pregnancy is established after IVF and PID, the additional corrective measure might be more acceptable or even attractive to parents. However, whole genome screening - which would be needed to detect all possible diseases causing de novo mutations – is still costly (15,000 dollars in a state-of-the-art research laboratory according to a 2014 study), and the reproducibility and validity of the results is also rather poor [45, 46]. This practice entails a risk of false-positive results, leading to unwarranted gene correction. In clinical practice in the near future, diagnosis may therefore limit the potential of germline editing and possibly allow decisions to be made about which mutations should be screened (technology allows reliable detection of specific mutations, but not every possible mutation). This parent-centered view also needs to be considered when decisions are made about legalizing therapeutic options; it also requires special education for future clinicians and patient guidance service staff.

How to take into account previous discussions in the CRISPR/Cas9 debate Up to this point, this article has taken the route of separating facts from fiction [82] as one means of simplifying the discussion and identifying key regulatory aspects and ethical issues that will become relevant in the near future. For example, while it is highly improbable that intelligence enhancement will be achieved, bodily enhancement may very well be. CRISPR/Cas9 may make it much easier to develop certain somatic gene therapies, while others will not be affected very much by this technological revolution. In general, a nuanced assessment of technical feasibility and an ethical justification of different applications is needed, e.g. concerning the issue of the just allocation of resources. Another important step is to identify value- and interest-laden assumptions and arguments based upon them in order to reach decisions that come closer to an ideal consensus and include all the stakeholders in society with their respective values. This was done in a

fairly approximate way by analyzing the differences in framing, comparators used and distinctions between stakeholder groups and by speculating about the possible underlying interests and values. Researchers are supposed to be interested first and foremost in conducting their research and might draw a distinction between application-oriented and basic research, fearing that the latter could be restricted due to moral objections that arise primarily from the former. For similar reasons, a patent-holder or pharmaceutical company developing somatic gene therapies based on CRISPR/Cas9 might underline the differences in terms of safety and ethics between germline therapy and somatic therapy. It is also natural for distinctions of this kind to be made in the context of media coverage, as they simplify the discussion, but they should at least be avoided by decision makers. While it may be attractive to draw lines between different kinds of research or application purposes such as enhancement and therapy, where to draw the line is not an easy question, nor one that is likely to be most important right now. Framing always means that there is a bias towards or neglecting of certain aspects, though it can also be positively viewed as offering different perspectives that could be combined to obtain a comprehensive picture of the ethical implications of CRISPR/ Cas9. However, stakeholder perspectives should be weighed in a representative fashion that reflects the values and interests of society as a whole, and especially of those citizens who pay for research and treatments and of those patients who are personally affected. Carefully chosen comparators may prove a key way of learning from previous discussions, by comparing not only the technologies themselves but also the debates about them. An evaluation of the scientific state of the art and ELSA can be performed with respect to an existing technology in an attempt to identify new ethical issues. Based on the result of this comparison, arguments from previous and ongoing debates can be compared in order to identify changes in values. It is of course difficult to ascertain the precise influence of facts and values on the prominence of arguments. Did the fact of the first IVF child and improvements in genetic diagnostics change the public’s acceptance of PID, or was it rather a question of the changes to the value ascribed to naturalness on the one hand and health or the possibility to have a child that is genetically one’s own on the other? Probably the two aspects go hand in hand. Historical perspectives [67, 70] are of course only case studies with limited predictive value, but they could

Nanoethics

at least provide some insights into the possible response of regulatory bodies faced with an emerging technology. Distinctions, frames and comparators are dependent on one other. For example, the distinction between enhancement vs. therapy and the comparator of the Asilomar conference are clearly framed in ethics and risk, whereas a socio-economic frame would make less sense. However, a comparator may also be used in different frames. In the remainder of this article, three examples will be given to illustrate the challenges and benefits of using different comparators and frames.

Mitochondrial transfer as a comparator for CRISPR/Cas9-germline editing Mitochondrial transfer is a relatively novel technique for treating certain diseases that are caused by mutations in the mitochondrial DNA, a tiny part of the total genome that is located in small organelles called mitochondria. It was approved as a therapy in the UK by the Human Fertilisation and Embryology Authority (HFEA), but only for specialized licensed clinics. It can be described as a germline modifying technology, and like germline editing may benefit a small number of affected people. This comparator is therefore relatively obvious to patients and clinicians, but is avoided by some scientists who do not want to reduce CRISPR/Cas9 applications to germline therapy and explicitly refuse to comment on mitochondrial transfer and the decision of the HFEA to allow this procedure [9]. One focus here will be on the available data concerning risk and ethical issues that might be compared, though another option would for example be to compare socio-economic aspects of the two procedures. The challenge of making the right diagnosis and therapy decisions as discussed above is already familiar from the field of mitochondrial replacement. In reproduction, mitochondria go through a bottleneck, that is to say only a small proportion of the several hundred or thousand mitochondria of the mother, all of which have a slightly different genome, will be passed on to the child. Furthermore, the resulting phenomenon, known as mosaicism, may occur during development of the embryo in utero, resulting in different mitochondrial genome compositions in different body cells. It is therefore difficult to predict the risk of transmission of heteroplasmic diseases, which occur when the proportion of mitochondria with an erroneous genome exceed

a certain threshold [75]. Moreover, only one out of every 40 people with abnormal mitochondrial DNA (mtDNA) requires therapy; the rest are asymptomatic or show only mild symptoms [75]. When we look at the possible unintended consequences of both types of germline modification, concerns arise about the incompatibility of donor mitochondrial and recipient nuclear DNA on the one hand [76] and off-target effects and mosaicism for genome editing on the other. In the case of genome editing, knowledge of the Bright^ modifications is crucial in order not to do more harm than good, whereas mitochondrial transfer does not require any deeper knowledge about the exact mutation that causes the disease, because the whole mtDNA from a healthy person is transferred. When we compare evidence from animal studies, there is a study of monkeys (now several years old) [77] which were born with donated mitochondrial DNA that failed to show any abnormalities in terms of development or health [76]. Genome editing of the germline has been tried in both monkeys and humans, in both instances with less encouraging results that might have been expected considering the preceding experiments on mice (for an overview of experiments in different species see [23]). To defend the general prospects of germline editing, some scientists have stated that the best available techniques may not have been used so far [8]. The problem of both techniques is that harmful side effects in clinical trials would be irreversible. Consequently, interviewees (clinicians, reproductive medicine experts) in a study on mitochondrial replacement stated that the desire to have a child that is genetically one’s own – which is the essential benefit of this technique and one that cannot be achieved by alternative techniques - does not legitimate experimental methods [47]. Taken together, theoretical considerations and experimental studies suggest that germline editing is technically more demanding, and that results from animal studies may prove less transferable to humans than is the case with mitochondrial replacement. On the other hand, disease-causing mutations in the nuclear genome are easier to detect and to select embryos against than is the case with mtDNA mutations. A comparison is not unambiguous even at the technology level and thus does not lead to any clear answer about which technique is more acceptable in terms of risk. However, in view of the debates on both techniques, it seems that it may well become more difficult in future

Nanoethics

to justify a general ban on germline editing for safety reasons once technical improvements have been achieved. Though CRISPR/Cas9 germline editing has been described as the latest step on the provocation ladder, it is doubtful whether a point has been reached where a medical intervention will be banned simply on fundamental moral grounds. Reactions to mitochondrial transfer appear to have been very similar to reactions to human embryo experiments with CRISPR/Cas9 [78]. After monkey experiments had been reported, mitochondrial transfer was regarded as a slippery slope towards eugenics, as healthy donor mitochondria would not only protect against diabetes and Alzheimer’s but could also boost athleticism. Furthermore, it was regarded as too risky to attempt in humans. A comparison of mitochondrial transfer and germline editing thus reveals that the most serious issues regarding CRISPR/ Cas9 and human germline therapy are already present with regard to a technique that will soon be applied in clinical practice. In principle, mitochondrial transfer may be used for enhancement purposes [79], is a heritable modification of the genome, challenges traditional clinical trial procedures [76], and may be seen as a generally very costly procedure to treat certain infertility conditions. One may argue that germline genome editing nevertheless raises more serious concerns than mitochondrial transfer due to its higher versatility and technical differences in the way treatment is carried out. However, any ranking of ethical acceptability leaves the question of absolute standards or ‘red lines’ unresolved. One may challenge the HFEA’s decision to allow mitochondrial replacement rather than supposing that all ethical issues are resolved. In this regard, a detailed comparison of ethical and safety issues may show that the breaking of taboos is by no means new, and has happened with every new assisted reproduction technique. As soon as medical benefits are proven, acceptance can increase rapidly and turn a practice that formerly broke taboos into daily clinical routine, as was already seen during the history of IVF [67] and may happen again in the near future with mitochondrial replacement. Two things can be learned from this comparison. First, that CRISPR does not lead to any fundamentally new ethical issues, and second, that the outcome of a comparison with a previous, already established technology is neither straightforward nor entirely objective. It may rather result in different assessments of the degree of similarity, depending on which aspects are the

focus. While both techniques alter the germline, for example, they actually affect different kinds of genomes upon closer inspection [77]. Both may be used for enhancement, and although mitochondrial transfer may affect only a limited range of human traits it is already technically feasible and approved for therapeutic use in the UK. Differences concerning the seriousness of ethical implications can likewise be judged and interpreted in different ways. If the two techniques are considered to be quite similar, it could follow that germline editing should be allowed given that mitochondrial transfer has already been allowed by the UK ethics council. However, it could also be concluded that this decision was wrong on the basis of the assumption that germline editing is wrong. If the two techniques are considered to be quite different, i.e. if germline editing is judged to have much more severe ethical implications and safety issues, then mitochondrial transfer might appear relatively harmless and uncontroversial as compared with its successor. This might indeed be considered as a slippery slope, when subsequent technologies steal attention from previous ones that have not been properly discussed.

Philosophical and psychological framing: From unnaturalness to knowledge-capability imbalance Though the argument of unnaturalness - which defines germline modification as being morally wrong because it violates some kind of natural order – is discussed mostly in bioethical literature and may in some respects be too detached from reality, it should perhaps be considered carefully for the following reasons. On the one hand, the naturalness argument may be misused to rationalize an irrational psychological aversion or fear of the unknown, qualifying the BYuk!^ factor or Bthe wisdom of repugnance^ as intuitive ethics without the need for further justification [80]. On the other hand, one of the sources of these aversive feelings towards modifying the human body are myths such as the stories of Frankenstein or Daedalus, which make reference to moral obligations and taboos and warn against hubris. Such warnings may not be irrational in the case of CRISPR/Cas9: first, in light of the rather sobering results of the germline editing experiments in man and monkeys and given the uncertainties of technical knowledge transfer from animal tests to human applications, and second, with regard to the complexity of the

Nanoethics

genome, its influence on the development of the human body and interactions with the environment, which have long been underestimated [80]. Our capabilities may proceed faster than our knowledge about the exact outcomes of a new technology’s application. Though some argue against it [38], this situation appears to justify the application of a precautionary principle, especially in genomic medicine. Science has demonstrated that the human genome is more complex than it was thought to be two decades ago, yet the hope that we will be able to take control of our genes still predominates, and at times is also hyped. The mass media talk about man steering his own evolution [29], while pharmaceutical companies strongly promote the idea of personalized medicine that is tailored to the genetics of individuals, yet most scientists are much more modest in their claims and recognize that we do not know enough to apply CRISPR/Cas9 germline editing in a sound way [8]. Overestimating our knowledge may lead to technically feasible interventions having harmful outcomes. As already mentioned, uncertainty and risk are separate concepts. When it is possible to estimate risks, for example to calculate the probability of harm, emotions may lead to biased views when numbers are ignored. If unknowns are a threat, however, rationality is not necessarily superior in decision making and emotions could perhaps protect people from engaging in risky behavior. Emotional repulsion can also be seen in a positive light, as it may stop thoughtless actions and encourage people to consider rational arguments that are more than merely a rationalization of irrational objections. Another critical point is that replacing parts of the natural reproduction process with technological and commercial substitutes results in a situation in which Bdeception, secrecy, abuse and manipulation [are] almost inevitable^, as Oderberg and Laing pointed out, perhaps somewhat overly pessimistically [81]. Accordingly, it is not CRISPR/Cas9 germline editing per se that would be unethical on account of its unnaturalness; rather the necessary social and commercial structures surrounding it would pose a moral threat. Privatization of health care, competition for research funds, and the style of mass media coverage may indeed further exacerbate or contribute to ethical issues associated with the use and application of CRISPR/Cas9. Intentionally or not, the debate may be channeled into certain directions preferred by stakeholders, thereby obscuring relevant issues, and commercial interests may overshadow ethical awareness and guide decisions. The first companies

seeking to use CRISPR/Cas9 commercially were launched shortly after the first experiments on human embryos. As was the case with antibody therapies, it would be easy to suspect that companies developing gene therapies will make efforts to emphasize the benefits rather than the potential risks of germline editing. They may also choose not to use an economic frame if the cost/benefit ratio of these therapies turns out to be poor. More research is needed to find out how companies affect public awareness of ethics, usefulness and acceptance of germline editing, as well as the decisions of ethics committees and clinicians. Stakeholder interests such as those of the pharma industry need to be carefully monitored in order to protect patients and make the best possible use of the health care budget without constraining innovations.

CRISPR/Cas9-germline editing – a historical frame A historical perspective might help one obtain a better idea of the possible impacts of emerging medical technologies in a societal and economic context. Although IVF was characterized at its outset by a lack of funding due to mostly ethical concerns, research proceeded with private money and led to the first IVF child in 1978 [67]. Since then, two million children have been born thanks to this procedure, and the technology has evolved considerably, resulting in better access and efficiency as well as a broader spectrum of indications [70]. This therapy is made accessible for example not only to women who lack the reproductive organs necessary to become pregnant naturally, but also to older women who wish to have a child after pursuing a successful professional career [70]. Will germline editing be privately funded, will it succeed, will it become accepted, and will it be used for an ever wider range of therapeutic and non-therapeutic purposes thanks to technological improvements? Is such a course of events inevitable or can it be steered, and if so, how should this happen and with what justification? Though history naturally does not allow one to predict the future, it may at least give certain strong indications that acceptance can be reached even in cases where ethical concerns prevail or become even more severe. IVF was a prerequisite for the selection of genetically screened embryos in vitro, and the genetic screening of embryos is a prerequisite for the germline modification of diseases that the parents do not have. In the past, technological developments and the

Nanoethics

way they were combined led to a broader spectrum of indications for assisted reproduction. Will this trend continue? This question has yet to be answered, and judging this possible future is another issue which should be based on a thorough analysis of the societal and medical outcomes of IVF, for which data from 40 years of practice is available today.

Summary and conclusions In contrast to the theoretical discussions to be found in bioethics literature and the somewhat speculative presentations of the debate by the mass media, this article sought to present a more realistic and context-based technology assessment view of germline editing and other therapeutic applications of CRISPR/Cas9. A scenario based on the technological state of the art and the social and legal framework is needed to separate facts from fiction in order not to waste resources on the ethical reflection of the latter [82], reduce the complexity of the discussion and enable the responsible development and handling of an emerging technology like CRISPR/Cas9. From a fairly pragmatic viewpoint, theoretical arguments against germline editing, however convincing or at least consistent they may be, are unlikely to have any major impact on actual decisions in the long run, as such arguments have been around for 50 years and were not able to prevent the legalization of IVF, PID or, more recently, mitochondrial transfer. Although ethical concerns will always remain, acceptance may nevertheless increase as the first medical success of germline editing in humans becomes apparent. The application of germline editing appears almost inevitable given the existing therapeutic justification, albeit for only very few patients, and its rapidly evolving technology. Bioethics needs to focus on the clinic and the needs and feelings of patients if it is to be able to propose ethical solutions to practical problems. Clinical guidelines, laws and consultation that help parents make decisions are needed rather than insufficiently nuanced views of the CRISPR/Cas9 technology and calls for a general ban. One factor that could be more pressing today than was the case in recent decades might be the economic aspects of sophisticated technologies in medicine and the issue of just distribution of resources in the health care system.

A comprehensive account of all the ethical and other pro and con arguments that have been brought forward with regard to CRISPR/Cas9 germline editing constitutes only a first step, and may in itself prove more confusing than helpful when it comes to decision making, be it by policy makers, clinicians or patients. The complexity of the issues surrounding mitochondrial transfer is illustrated by a study of the German ethics council [83]. Rather than answering the question: BShould mitochondrial transfer be allowed?^ it offers a complicated decision tree with numerous different branches that have to be taken into account and weighed against each other in order to reach a decision. However, the ethical judgement and allowance of CRISPR/Cas9 applications in humans will depend eventually on a variety of subjective values and moral assumptions. Secular bioethics cannot provide definitive answers about the justification of these values and assumptions in a postmodern world [84]. Bioethics can however offer an understanding of the different moral perspectives on bioethical issues [84]. At times these are hidden or unknown, giving rise to a situation in which discussants end up Btalking at cross purposes^ [85]. An analysis of existing frames and comparators and the additional use of unorthodox or less dominant comparators, e.g. stories about human hubris, existing assisted reproduction techniques, or antibody therapy, could open up philosophical, psychological, historical or socio-economic frames that would enrich the discussion.

Outlook: Public participation, internationality and the role of bioethics It was pointed out that dichotomies have been established and predominate in discussions of germline editing due to certain assumptions about public opinion, though this could not be confirmed by surveys [86]. For example, most respondents did not make a clear distinction between somatic and germline therapy, and eugenics was not seen as such an important ethical issue with regard to gene therapy [86]. An assessment of both values and knowledge has been undertaken via online surveys more recently, with interesting results that suggest that the media has a major impact when it comes to increasing acceptability even where ethical concerns remain, and that showed that public concerns and bioethical literature sometimes diverge [87, 88].

Nanoethics

Bioethics has been sharply criticized recently for being too detached from reality and for arguing about theoretical principles of dignity, autonomy and the like when it comes to alleviating suffering from disease. Instead of being overly restrictive, bioethics would do better to rapidly assess evolving new technologies in context and on a case-by-case basis [89, 90]. Bioethicists could give consideration to ELSA and a differentiated view of technologies and by taking sides with the ‘silent’ voices that policy makers are not yet hearing, following the trend of participatory approaches in the ethics of genomics [60]. Furthermore, the international and intercultural discussion is of great importance in an age where new technological developments are not limited by borders, while national regulatory differences constitute an additional complicating factor for bioethics. As the director general of UNESCO stated, there is a Bneed for people of all nationalities and their governments to look beyond their borders in understanding the bioethical issues that are being generated and in providing solutions that are fair to all and compatible with the plurality of values and interests in the international community^ [60]. International harmonization of the relevant laws would prevent gene therapy tourism to less strictly regulated nations, but should also be based on an international consensus on values [86]. This article attempted to illustrate the wide range of issues and perspectives that need to be taken into account when ethical discussions are supposed to be relevant to reality and able to influence decisions for the good of society as a whole. It makes no claim to be comprehensive, but rather sought to outline an agenda for bioethics, and especially for collaboration between disciplines, that could prove fruitful for the discussion of CRISPR/Cas9 genome editing in humans. Bioethics has to be aware of social and legal contexts, take a nuanced view of emerging technologies, and listen to the public. Furthermore, international perspectives are needed in a globalized scientific world. We are now also in a position to view assisted reproduction medicine from a historical perspective given that the first IVF child was born almost 40 years ago. This may be a demanding task, yet the tough reality is that this is what we need to accomplish when it comes to the hyped CRISPR/Cas9 technology. However, there are enough promising efforts that give rise to considerable hope that bioethics can underline its important role and answer critical voices.

Compliance with ethical standards Conflict of Interest The author declares that she has no conflict of interest.

References 1. Liang P, Xu Y, Zhang X, Ding C, Huang R, Zhang Z, Lv J, Xie X, Chen Y, Li Y, Sun Y, Bai Y, Songyang Z, Ma W, Zhou C, Huang J (2015) CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell 6(5):363–372. doi:10. 1007/s13238-015-0153-5 2. Doudna JA, Charpentier E (2014) The new frontier of genome engineering with CRISPR-Cas9. Science 346(6213):1077. doi:10.1126/science.1258096 3. Cox DBT, Platt RJ, Zhang F (2015) Therapeutic genome editing: prospects and challenges. Nat Med 21(2):121–131. doi:10.1038/nm.3793 4. Gersbach CA (2014) Genome engineering: the next genomic revolution. Nat Methods 11(10):1009–1011. doi:10.1016/j. tibtech.2013.04.004 5. Mussolino C, Mlambo T, Cathomen T (2015) Proven and novel strategies for efficient editing of the human genome. Curr Opin Pharmacol 24:105–112. doi:10.1016/j.coph.2015. 08.008 6. Singh P, Schimenti JC, Bolcun-Filas E (2015) A mouse geneticist’s practical guide to CRISPR applications. Genetics 199(1):1–15. doi:10.1534/genetics.114.169771 7. Ran Le Cong FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823. doi:10.1126/science.1231143 8. Bosley KS, Botchan M, Bredenoord AL, Carroll D, Charo RA, Charpentier E, Cohen R, Corn J, Doudna J, Feng G, Greely HT, Isasi R, Ji W, Kim JS, Knoppers B, Lanphier E, Li J, Lovell-Badge R, Martin GS, Moreno J, Naldini L, Pera M, Perry ACF, Venter JC, Zhang F, Zhou Q (2015) CRISPR germline engineering—the community speaks. Nat Biotechnol 33(5):478–486. doi:10.1038/nbt.3227 9. Lanphier E, Urnov F, Haecker SE, Werner M, Smolenski J (2015) Don’t edit the human germ line. Nature 519(7544): 410–411. doi:10.1038/519410a 10. Vogel G (2015) Embryo engineering alarm. Science 347(6228):1301. doi:10.1126/science.347.6228.1301 11. Norman C (1983) Clerics urge ban on altering germline cells. Science 220(4604):1360–1361 12. Torgersen H, Schmidt M (2013) Frames and comparators: how might a debate on synthetic biology evolve? Futures 48(100):44–54. doi:10.1016/j.futures.2013.02.002 13. AJ Newson, A Wrigley (2015) Identifying key developments, issues and questions relating to techniques of genome editing with engineered nucleases. Background paper. http:// nuffieldbioethics.org/wp-content/uploads/Genome-EditingBriefing-Paper-Newson-Wrigley.pdf. Accessed 1 Dec 2015 14. Sarewitz D (2015) CRISPR: science can’t solve it. Nature 522(7557):413–414. doi:10.1038/522413a

Nanoethics 15. Ledford H (2015) CRISPR the disruptor. Nature 522(7554): 20–24. doi:10.1038/522020a 16. Kaiser J, Normile D (2015) Embryo engineering study splits scientific community. Science 348(6234):486–487. doi:10. 1126/science.348.6234.486 17. Pollack R (2015) Eugenics lurk in the shadow of CRISPR. Science 348(6237):871. doi:10.1126/science.348.6237.871-a 18. Miller HI (2015) Germline gene therapy: we’re ready. Science 348(6241):1325. doi:10.1126/science.348.6241.1325-a 19. Krishan K, Kanchan T, Singh B (2016) Human genome editing and ethical considerations. Sci Eng Ethics 22(2): 597–599. doi:10.1007/s11948-015-9675-8 20. Goldim JR (2015) Genetics and ethics: a possible and necessary dialogue. J Community Genet 6(3):193–196. doi:10. 1007/s12687-015-0232-6 21. Sugarman J (2015) Ethics and germline gene editing. EMBO Rep 16(8):879–880. doi:10.15252/embr.201540879 22. Carroll D, Charo RA (2015) The societal opportunities and challenges of genome editing. Genome Biol 16(1):242. doi: 10.1186/s13059-015-0812-0 23. Araki M, Ishii T (2014) International regulatory landscape and integration of corrective genome editing into in vitro fertilization. Reprod Biol Endocrinol 12:108. doi:10.1186/14777827-12-108 24. Ishii T (2015) Germline genome-editing research and its socioethical implications. Trends Mol Med 21(8):473–481. doi:10.1016/j.molmed.2015.05.006 25. Merlot J (2015) Umstrittene Experimente: Forscher manipulieren Erbgut menschlicher Embryonen. SPIEGEL, 24 April 2015. http://www.spiegel.de/wissenschaft/medizin/ forscher-manipulieren-gene-menschlicher-embryonen-a1030142.html. Accessed 1 Dec 2015 26. Focus (2015) Forscher aus China verändern Erbgut von Embryos. Focus, 24 April 2015. http://www.focus.de/ wissen/mensch/tabubruch-der-gentechnik-chinesischeforscher-veraendern-erbgut-von-embryos_id_4635723.html. Accessed 1 Dec 2015 27. Schlütter J (2015) Tagesspiegel, 24 April 2015. Die Weltgemeinschaft sollte über ethische Grenzen diskutieren! http://www.tagesspiegel.de/wissen/gentechnisch-optimierteembryonen-dieweltgemeinschaft-sollte-ueber-ethischegrenzen-diskutieren/11681026.html. Accessed 1 Dec 2015 28. Zinkant K (2015) Eine Grenze ist überschritten. Süddeutsche Zeitung, 25 April 2015. http://www.sueddeutsche.de/wissen/ genetische-manipulation-eine-grenze-ist-ueberschritten-1. 2452395. Accessed 1 Dec 2015 29. Bahnsen U (2015) Der Mensch kann seine Evolution nun selbst bestimmen. ZEIT, 23 April 2015. http://www.zeit.de/ wissen/gesundheit/2015-04/genetik-erbgut-embryo-china. Accessed 1 Dec 2015 30. Zimmer C (2015) Editing human embryos: so this happened. http://phenomena.nationalgeographic.com/2015/04/22/ editing-human-embryos-so-this-happened. Accessed 1 Dec 2015 31. Economist (2015) Editing Humanity. Economist, 22 August 2015. http://www.economist.com/news/leaders/21661651new-technique-manipulating-genes-holdsgreat-promise-rulesare-needed-govern-its. Accessed 1 Dec 2015 32. Baltimore D, Berg P, Botchan M, Carroll D, Charo RA, Church G, Corn JE, Daley GQ, Doudna JA, Fenner M, Greely HT, Jinek M, Martin GS, Penhoet E, Puck J,

33.

34.

35.

36.

37. 38.

39.

40.

41.

42. 43.

44. 45.

46.

47.

48. 49.

Sternberg SH, Weissman JS, Yamamoto KR (2015) A prudent path forward for genomic engineering and germline gene modification. Science 348(6230):36–38. doi:10.1126/ science.aab1028 Berg P (2008) Meetings that changed the world: Asilomar 1975: DNA modification secured. Nature 455(7211):290– 291. doi:10.1038/455290a Kaebnick GE (2015) A Moratorium on Gene Editing? http:// www.thehastingscenter.org/Bioethicsforum/Post.aspx?id= 7359&blogid=140. Accessed 1 Dec 2015 Heavey P (2013) Synthetic biology ethics: a deontological assessment. Bioethics 27(8):442–452. doi:10.1111/bioe. 12052 Ter Meulen R, Biller-Andorno N, Newson A, Hunter D (2013) How to object to radically new technologies on the basis of justice: the case of synthetic biology. Bioethics 27(8):426– 434. doi:10.1111/bioe.12049 Smith K (2013) Synthetic biology: a utilitarian perspective. Bioethics 27(8):453–463. doi:10.1111/bioe.12050 Smith KR, Chan S, Harris J (2012) Human germline genetic modification: scientific and bioethical perspectives. Arch Med Res 43(7):491–513. doi:10.1111/bioe.12050 Pugh J (2015) Autonomy, natality and freedom: a liberal reexamination of habermas in the enhancement debate. Bioethics 29(3):145–152. doi:10.1111/bioe.12082 Sunkara SK, Rittenberg V, Raine-Fenning N, Bhattacharya S, Zamora J, Coomarasamy A (2011) Association between the number of eggs and live birth in IVF treatment: an analysis of 400 135 treatment cycles. Hum Reprod 26(7):1768–1774. doi: 10.1093/humrep/der106 Das SK, Menezes ME, Bhatia S, Wang X-Y, Emdad L, Sarkar D, Fisher PB (2015) Gene therapies for cancer: strategies, challenges and successes. J Cell Physiol 230(2):259–271. doi:10.1002/jcp.24791 Ledford H (2015) Mini enzyme moves gene editing closer to the clinic. Nature 520(7545):18. doi:10.1038/520018a Kendler KS (2013) What psychiatric genetics has taught us about the nature of psychiatric illness and what is left to learn. Mol Psychiatry 18(10):1058–1066. doi:10.1038/mp.2013.50 Lander ES (2015) Brave new genome. N Engl J Med 373(1): 5–8. doi:10.1056/NEJMp1506446 Powledge TB (2014) Whole-genome sequencing in your doctor’s office? A reality check, but sooner than later. http://www. geneticliteracyproject.org/2014/03/25/whole-genomesequencing-in-your-doctors-office-a-reality-check-butsooner-than-later/. Accessed 1 Dec 2015 Winand R, Hens K, Dondorp W, de Wert G, Moreau Y, Vermeesch JR, Liebaers I, Aerts J (2014) In vitro screening of embryos by whole-genome sequencing: now, in the future or never? Hum Reprod 29(4):842–851. doi:10.1093/humrep/ deu005 Hens K, Dondorp W, de Wert G (2015) A leap of faith? An interview study with professionals on the use of mitochondrial replacement to avoid transfer of mitochondrial diseases. Hum Reprod 30(5):1256–1262. doi:10.1093/humrep/dev056 Hammitt JK (2002) QALYs versus WTP. Risk Anal 22(5): 985–1001 EMA, Guideline on the quality, non-clinical and clinical aspects of gene therapy medicinal products: Draft. http://www. ema.europa.eu/docs/en_GB/document_library/Scientific_ guideline/2015/05/WC500187020.pdf. Accessed 1 Dec 2015

Nanoethics 50. U.S. Department of Health and Human Services- FDACenter for Biologics Evaluation (2006) Gene therapy clinical trials - observing subjects for delayed adverse events: guidance for industry. Accessed 1 Dec 2015 51. Europäisches Parlament und Europäischer Rat (2009) RICHTLINIE 2009/120/EG, 2009 52. Knight FH (1921) Risk, uncertainty and profit. Hart, Schaffner & Marx, Houghton Mifflin, Boston, MA 53. DACEHTA-Danish Centre for Health Technology Assessment (2015) Health technology assessment handbook. http://www.ema.europa.eu/docs/en_GB/document_library/ Scientific_guideline/2015/05/WC500187020.pdf. Accessed 1 Dec 2015 54. Nature Medicine (2015) Germline editing: time for discussion. Nat Med 21(4):295. doi:10.1038/nm.3845 55. Morrison C (2015) $1-million price tag set for glybera gene therapy. Nat Biotechnol 33(3):217–218. doi:10.1038/ nbt0315-217 56. ECDC & EMEA (2009) The bacterial challenge - time to react: a call to narrow the gap between multidrug-resistant bacteria in the EU and development of new antibacterial agents, 2009. Accessed 1 Dec 2015 57. Kuhrt N (2013) Was darf ein Monat Leben kosten? Frankfurter Allgemeine Zeitung, 20 January 2010. http:// www.faz.net/aktuell/wissen/medizin/krebstherapie-was-darfein-monat-leben-kosten-1907448.html. Accessed 1 Dec 2015 58. Wirth T, Parker N, Ylä-Herttuala S (2013) History of gene therapy. Gene 525(2):162–169. doi:10.1016/j.gene.2013.03. 137 59. Clinicaltrials.gov. Accessed 1 Dec 2015 60. Knoppers BM, Chadwick R (2005) Human genetic research: emerging trends in ethics, nature reviews. Genetics 6(1):75– 79. doi:10.1038/nrg1505 61. Cressey D, Abbott A, Ledford H (2015) UK scientists apply for licence to edit genes in human embryos. http://www. scientificamerican.com/article/scientists-apply-for-license-toedit-genes-in-human-embryos. Accessed 8 Dec 2015 62. Tu Z, Yang W, Yan S, Guo X, Li X-J (2015) CRISPR/Cas9: a powerful genetic engineering tool for establishing large animal models of neurodegenerative diseases. Mol Neurodegener 10:35. doi:10.1186/s13024-015-0031-x 63. Combes RD, Balls M (2014) Every silver lining has a cloud: the scientific and animal welfare issues surrounding a new approach to the production of transgenic animals. ATLA Altern Lab Anim 42(2):137–145 64. Reardon S (2015) NIH reiterates ban on editing human embryo DNA. http://www.nature.com/news/nih-reiterates-ban-onediting-human-embryo-dna-1.17452. Accessed 8 Dec 2015 65. Fateh-Moghadam B (2011) Rechtliche Aspekte der somatischen Gentherapie. In: Fehse B, Domasch S (eds) Gentherapie in Deutschland. Dornburg: Wissenschaftlicher Verlag, pp 151–184 66. Reich J (ed) (2015) Genomchirurgie beim Menschen zur verantwortlichen Bewertung einer neuen Technologie: eine Analyse der interdisziplinären Arbeitsgruppe Gentechnologiebericht. BBAW, Berlin 67. Johnson MH, Franklin SB, Cottingham M, Hopwood N (2010) Why the medical research council refused Robert Edwards and Patrick Steptoe support for research on human conception in 1971. Hum Reprod 25(9):2157–2174

68. Bundesministerium für Gesundheit. PID. http://www.bmg. bund.de/glossarbegriffe/p-q/praeimplantationsdiagnostik. html. Accessed 1 Dec 2015 69. van Montfoort APA, Hanssen LLP, de Sutter P, Viville S, Geraedts JPM, de Boer P (2012) Assisted reproduction treatment and epigenetic inheritance. Hum Reprod Update 18(2): 171–197 70. Wang J, Sauer MV (2006) In vitro fertilization (IVF): a review of 3 decades of clinical innovation and technological advancement. Ther Clin Risk Manag 2(4):355–364 71. Karen Geraghty, Protecting the Public: Profile of Dr. Frances Oldham Kelsey. http://journalofethics.ama-assn.org/2001/07/ prol1-0107.html. Accessed 1 Dec 2015 72. Review of scientific methods to avoid mitochondrial disease (2014) http://www.hfea.gov.uk/8807.html. Accessed 1 Dec 2015 73. Europäisches Parlament und Rat zur Schaffung eines Gemeinschaftskodexes für Humanarzneimittel im Hinblick auf Arzneimittel für neuartige Therapien, zur Änderung der Richtlinie 2001/83/EG des Europäischen Parlaments und des Rates zur Schaffung eines Gemeinschaftskodexes für Humanarzneimittel im Hinblick auf Arzneimittel für neuartige Therapien: RICHTLINIE 2009/120/EG, 2009. 74. Asher Mullard (2015) Use of personalized cancer drugs runs ahead of the science. http://www.nature.com/news/use-ofpersonalized-cancer-drugs-runs-ahead-of-the-science-1. 18389. Accessed 1 Dec 2015 75. Nuffield Council (2012) Novel techniques for the prevention of mitochondrial DNA disorders: an ethical view. Nuffield Council on Bioethics, London 76. Mitalipov S, Wolf DP (2014) Clinical and ethical implications of mitochondrial gene transfer. Trends Endocrinol Metab 25(1):5–7. doi:10.1016/j.tem.2013.09.001 77. Tachibana M, Sparman M, Sritanaudomchai H, Ma H, Clepper L, Woodward J, Li Y, Ramsey C, Kolotushkina O, Mitalipov S (2009) Mitochondrial gene replacement in primate offspring and embryonic stem cells. Nature 461(7262): 367–372. doi:10.1038/nature08368 78. Jesse Reynolds (2009) Monkeys, mitochondria, and the human germline. http://www.thehastingscenter.org/ Bioethicsforum/Post.aspx?id=3904&blogid=140. Accessed 1 Dec 2015 79. Kouros N (2013) Eugenics concerns over mitochondrial replacement. Monash Bioeth Rev 31(2):5–6 80. Ball P (2014) The art of medicine: unnatural reactions. Lancet 383:1964–1965 81. Smajdor A, Ives J, Baldock E, Langlois A (2008) Getting from the ethical to the empirical and back again: the danger of getting it wrong, and the possibilities for getting it right. Health Care Anal 16(1):7–16. doi:10.1007/s10728-0070079-z 82. Nordmann A, Rip A (2009) Mind the gap revisited. Nat Nanotechnol 4(5):273–274. doi:10.1038/nnano.2009.26 83. Deutscher Ethikrat (2014) Sollten Vorkern- und Spindeltransfer bei mitochondrialen Erkrankungen in Deutschland zulässig sein? http://www.ethikrat.org/dateien/ pdf/jt-22-05-2014-vorkern-und-spindeltransfer.pdf. Accessed 1 Dec 2015 84. Engelhardt HT (2011) Confronting moral pluralism in posttraditional Western societies: bioethics critically reassessed. J Med Philos 36(3):243–260

Nanoethics 85. Patenaude J, Legault GA, Béland J-P, Parent M, Boissy P (2011) Moral arguments in the debate over nanotechnologies: are we talking past each other? NanoEthics 5(3):285–293. doi: 10.1007/s11569-011-0132-0 86. Macer D (1995) International perceptions and approval of gene therapy. Hum Gene Ther 6:791–803 87. Robillard JM (2015) Communicating in context: a priority for gene therapy researchers. Expert Opin Biol Ther 15(3):315– 318. doi:10.1517/14712598.2015.1001735 88. Robillard JM, Roskams-Edris D, Kuzeljevic B, Illes J (2014) Prevailing public perceptions of the ethics of gene therapy. Hum Gene Ther 25(8):740–746. doi:10.1089/ hum.2014.030 89. Madhusoodanan J (2015) Bioethics accused of doing more harm than good. Nature 524(7564):139

90. Pinker S (2015) The moral imperative for bioethics. https:// www.bostonglobe.com/opinion/2015/07/31/the-moralimperative-for-bioethics/JmEkoyzlTAu9oQV76JrK9N/story. html. Accessed 1 Dec 2015 91. Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, Kang Y, Zhao X, Si W, Li W, Xiang AP, Zhou J, Guo X, Bi Y, Si C, Hu B, Dong G, Wang H, Zhou Z, Li T, Tan T, Pu X, Wang F, Ji S, Zhou Q, Huang X, Ji W, Sha J (2014) Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 156(4):836–843. doi:10.1016/j.cell.2014. 01.027 92. Wu Y, Liang D, Wang Y, Bai M, Tang W, Bao S, Yan Z, Li D, Li J (2013) Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell 13(6):659–662. doi:10.1016/j. stem.2013.10.016