Vol. 9 issue 1 2019

Mar 052019
 
Spread the love

Genetic information is encoded in the deoxyribonucleic acid (DNA). In form of a long double-helix molecule, lo-cated in living cells, it governs most of the organisms traits. Explicitly, information from genes is used to form func-tional gene products such as proteins. This process of gene expression is used by all known forms of life on earth to generate the macromolecular machinery for life. Thus, it poses the fundamental level of how the genotype causes the phenotype, i.e. the composite of organisms’ observ-able characteristics. Genomic modification is a powerful tool to amend those characteristics. Reproductional and environmentally caused changes to the DNA is a substrate for evolution. In nature, those changes happen and may cause favourable or unfavourable changes to the phenotype, which allow the cell or organism to improve or reduce the ability to survive and reproduce, respectively.

In the first half of the 20th century, several methods to alter the genetic structure of cells were discovered, which include exposing it to heat, X-rays, UV-light, and chemicals1-4. A significant number of crop cultivated today were developed using those methods of traditional muta-genesis, an example of which is Durum wheat, the most prevalent wheat for pasta production. With traditional mu-tagenesis thousands of mutations are introduced at random within the DNA of the plant. A subsequent screening iden-tifies and separates cells with favourable mutations in their DNA, followed by attempts to remove or reduce possible unfavourable mutations in those by mutagenesis or cross-breeding.

As those methods are usually unspecific and complex, researchers have developed site-determined gene editing techniques, the most successful of which is the so called CRISPR/Cas9 method (clustered regularly interspaced short palindromic repeats). This method borrows from how bacteria defend viral invasion.6 When the bacterium detects virus DNA invasion, it forms two strands of RNA (single helix molecules), one of which contains a sequence that matches that of the invading virus DNA and is hence called guide RNA. These two RNAs form a complex with a Cas9 protein, which, as a nuclease enzyme, can cleave DNA. When the guide RNA finds the target in the viral genome, the RNA-Cas9 complex will lock to a short se-quence known as the PAM, the Cas9 unzippes the viral DNA to which the RNA will match. Cas9 then cleaves the viral DNA, forcing the cell to repair the DNA.6 As this repair process is error prone, it may lead to mutations that might disable certain genes, changing the phenotype. In 2012 and 2013 it was discovered that the guide RNA can be considerably modified for the system to work site-determined5, and that by modifying the enzyme it not only works in bacteria and archaea, but also in eukaryotes (plants and animals), respectively.7

Figure 1: CRISPR/Cas9 working principle.8

Research published since demonstrated the method’s poten-tial for RNA-programmable genome editing. Modifications can be made so during the repair an artificially designed DNA sequence pairs with the cleaved ends, recombines and replaces the original sequence, introducing new genes to the genome.11,12 The advantages of this technique over tra-ditional gene editing methods is multifold. It can act very targeted, i.e. site- and therefore gene-specific in any form of known life. It is comparatively inexpensive, simple enough to be conducted in basic labs, effective, and fast regarding preparation and realisation. The production of multiplex ge-netically modified mice, for instance, was reduced from up to two years to few weeks,9 as CRISPR/Cas9 has the unique advantage over earlier genome editing methods, that multi-plexable targeting is easily achieved by co-expressing Cas9 with multiple single-guide RNAs simultaneously. Conse-quently, within few years after its discovery, it evolved to be the routine procedure for genome modification of virtually all model plants and animals.

The availability of such a method evokes medical and botanical development interests. A plethora of possible medical applications are discussed and researched, among which is healing cancer or treating genetic disorders. For cancer research it is imaginable to induce a multitude of deliberate mutations to artificially form cells similar to can-cerous cell, study the caused modification to the cells, and thus learn to inhibit their reproduction or the original muta-tion. In the clinical research focus now are blood diseases or those related to haematopoietic cells, such as leukaemia, HBV, HIV, or haemophilia.13,14 This is because for the treatment of those diseases, the cells (blood cells or bone marrow) can be extracted from the body in a known way, their genome can be edited in vitro by the CRISPR/Cas9 method, and finally the cells can be reintroduced to the body. The advantage of the extraction is that no additional vector (agent to help finding the right cells in vivo) is re-quired, and the genomic modification can be controlled ex vivo. While the editing efficiency with CRISPR-Cas9 can be extremely high, the resulting cell population will be inherently heterogeneous, both in the percentage of cells that were edited and in the specific genotype of the edited cells. Potentially problematic for in vivo application is the bacterial origin of the endonuclease Cas9. A large portion of humans show humoral and cell-mediated immune re-sponses to the Cas9 protein complex,10 most likely because of prior infection with related bacteria.

Although clinical applications of CRISPR/Cas9 grab a lot of media attention, agricultural applications draw even more commercial interest. Prospects here are the faster, cheaper and more targeted development of crops than by traditional methods of mutagenesis, which are extremely more aggressive in comparison. The main aim is unchanged though: improve plants regarding yield, resistance to dis-eases or vermin, and resilience to aridity, heat, cold, humid-ity, or acidity.15,16 CRISPR/Cas9 is therefore considered an important method to ameliorate agricultural food produc-tion to feed the earth’s ever-growing human population.

Regulations of thusly modified products vary largely be-tween countries. While Canada considers such plants equal to not genetically modified if no transgene was inserted, the USA assesses CRISPR plants on a case by case basis, gauging whether the modification would have been possible by natural mutation. This way they chose to not regulate mushrooms that do not turn brown and maize with an al-tered starch contend. Last year the European court of justice ruled all CRISPR/Cas9 modified plants as genetically mod-ified organisms, reasoning that the risks of such a novel method are unknown, compared to traditional mutagenesis as an established method of plant breeding.

Instigated by genome editing in human-embryonic cells in 201518 a group of scientists called for a moratorium to dis-cuss the possible risks and impact of the wide usage of the CRISPR/Cas9 technology, especially when it comes to mu-tations in humans.19 On the 2015 International Summit on Human Gene Editing leading international scientists con-sidered the scientific and societal implications of genome editing. The discussed issues span clinical, agricultural and environmental applications, with most attention focused on human-germline editing, owing to the potential for this application to eradicate genetic diseases and, ultimately, to alter the course of evolution. Some scientists advise to ban CRISPR/Cas9 based human genomic editing research for the foreseeable future, whereas others favour a rapid progress in developing it.20 A line of argument of support-ers of the latter viewpoint is, that the majority of ethical concerns are effectively based on methodical uncertainties of the CRISPR/Cas9 method at its current status, which can be overcome only with extensive research. Those methodical uncertainties include possible cleavage at undesired sites of the DNA, or insertion of wrong sequences at the cleavage site, resulting in the disabling of the wrong genes or even the creation of new genetic diseases.

Whilst a total ban is considered impractical because of the widespread accessibility and ease of use of this technology,21 the summit statement says, that “It would be irresponsible to proceed with any clinical use of germline editing unless and until (i) the relevant safety and effi-cacy issues have been resolved . . . and (ii) there is broad societal consensus about the appropriateness of the pro-posed application.” The moral concerns about embryonic or germline treatment base on the fact that CRISPR/Cas9 not only would allow the elimination of genetic diseases, but also enable genetic human enhancement, from simple tweaks like eye colour or non-balding to severe modifica-tions relating bone density, muscular strength or sensory and mental capabilities.

Although most scientist echo the summit statement, in 2018 a biochemist claimed to have created the first genetically edited human babies, two twin sisters. After in vitro fertil-ization, he targeted a gene that codes for a protein that one HIV variant uses to enter cells, enforcing a kind of HIV immunity, which is a very rare trait among humans.22 His conduct was harshly criticised in the scientific community, widely condemned, and-after enormous public pressure-redoing forbidden by the responsible regulatory offices.

Ultimately the CRIPSR/Cas9 technology is a paramount example of real world societal implications of basic re-search and demonstrates researchers’ responsibilities. This also raises the question whether basic ethical schooling should be part of every researcher’s education.

— Alexander Kronenberg

Read more:

[1] K. M. Gleason (2017) “Hermann Joseph Muller’s Study of X-rays as a Mutagen”

[2] Muller, H. J. (1927). Science. 66 (1699): 84–87.

[3] Stadler, L. J.; G. F. Sprague (1936). Proc. Natl. Acad. Sci. U.S.A. US Department of Agriculture and Missouri Agricul-tural Experiment Station. 22 (10): 572–8.
[4] Auerbach, C.; Robson, J.M.; Carr, J.G. (March 1947). Sci-ence. 105 (2723): 243–7.

[5] M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, E. Charpentier. Science, 337, 2012, p. 816–821.
[6] R. Sorek, V. Kunin, P. Hugenholtz. Nature reviews. Micro-biology. 6, 3, (2008), p. 181–186.

[7] Cong, L., et al., (2013). Science. 339 (6121) p. 819–823.

[8] https://commons.wikimedia.org/wiki/File:GRNA-Cas9.png

[9] H. Wang, et al., Cell. Band 153, 4, (2013), S. 910–918.

[10] D. L. Wagner, et al., Nature medicine. (2018).

[11] O. Shalem, N. E. Sanjana, F. Zhang; Nature reviews. Genet-ics 16, 5, (2015), p. 299–311.

[12] T. R. Sampson, D. S. Weiss; BioEssays 36, 1, (2014), p. 34–38.

[13] G. Lin, K. Zhang, J. Li; International journal of molecular sciences 16, 11, (2015), p. 26077–26086.

Mar 052019
 
Spread the love

Dr. Roman Stilling

Disclaimer: The opinions, views, and claims expressed in this essay are those of the author and do not necessarily reflect any opinion whatsoever of the members of the editorial board. The editorial board further reserves the right not to be responsible for the correctness of the information provided. Liability claims regarding damage caused by the use of any information provided will therefore be rejected.

Roman Stilling graduated with a B.Sc. in Biosciences from the University of Mün-ster in 2008 and received a Ph.D. degree from the International Max Planck Re-search School for Neurosciences / University of Göttingen in 2013. Afterwards he joined the APC Microbiome Ireland in Cork, Ireland, as postdoctoral researcher. Since 2016 he is the scientific officer for for the information initiative “Tierver-suche verstehen”1, coordinated by the Alliance of Science Organisations in Germany.


Ethical concerns on using animals in biomedical research have been raised since the 19th century. For example, in England the “Cruelty to Animals Act” was passed in 1876 as a result of a debate especially on the use of dogs un-der inhumane conditions such as invasive physiological experiments or demonstrations without general anaesthe-sia. Interestingly, it was Charles Darwin who put in all his scientific and political gravitas to push for regulation by the law while at the same time providing highly differen-tiated argumentation towards using animals for advancing knowledge, especially in the quickly developing field of physiology 1,2. In an 1881 letter to a Swedish colleague he wrote:

“[. . . ]I fear that in some parts of Europe little regard is paid to the sufferings of animals, and if this be the case I should be glad to hear of legislation against inhumanity in any such country. On the other hand, I know that physiology cannot possibly progress except by means of experiments on living animals, and I feel the deepest conviction that he

who retards the progress of physiology commits a crime against mankind.”3

Animal research as a moral dilemma

In this letter Darwin succinctly summarized the ethical dilemma that is the core of the debate on using animals for research: whether we may cause harm to animals if it is necessary to advance science and medicine.

In fact, the ability to suffer is generally accepted as the sin-gle most morally relevant criterion when animals are con-sidered as subjects of moral worth. This reasoning is based on the philosophies of Jeremy Bentham who’s thoughts on this matter culminated in the aphorism: “The question is not, Can they reason? nor, Can they talk? but, Can they suffer?”4

Today, animal welfare legislation is based on this notion in most countries, which has fundamental consequences on how different species of animals are protected by these reg-ulations. For example, in the EU, only the use of animals within the taxonomical subphylum Vertebrata (i.e. verte-brates) are covered by the respective EU directive.5 More recently also the use of Decapoda (e.g. crayfish, crabs, lob-sters) and Cephalopoda (e.g. squids, octopuses) falls within this regulation since it is assumed that these animals have a complex enough nervous system to perceive pain and expe-rience suffering.

Most current legislation in industrialized countries ac-knowledges that animals (not exclusively, but especially those able to suffer) have intrinsic value and a moral sta-tus that is different from other biological forms of life such as plants, fungi or bacteria and inanimate matter. At the same time no country has established legislation that con-siders the moral status of any animal the same as the moral status of a human being – irrespective of the developmental state or status of health of that human being.

Together this reasoning has led to the appreciation, that leg-islation cannot reflect a general rule of “one size fits all”, but a compromise needs to be implemented, where ethical and scientific judgment for each individual experiment or study is made on a case-by-case basis.

Adherence to the 3R-principle is necessary but not suf-ficient for ethical justification of laboratory animal use

The moral dilemma of inflicting harm on animals to ad-vance knowledge and medical progress was addressed in more detail in 1959, when William Russell and Rex Burch published “The principles of humane experimental technique”, in which they formulated the now famous 3R-principle for the first time: Replace, reduce, refine.6. This principle acknowledges human benefit from animal exper-iments but provides a guideline to minimize suffering in animals: Only if there is no alternative method to achieve the scientific goal, all measures to reduce the necessary number of animals in a given study, and the best possible conditions to confine suffering to the necessary minimum have been established, an experiment can be considered as potentially ethically justifiable. Meeting the 3R criteria is, however, a necessary but not sufficient requirement for eth-ical justification of a particular experiment.

Today the 3R-principle is well accepted worldwide7 as a formula to minimize animal suffering and has become an integral part of EU animal welfare regulations, which have been translated to national law in all EU member states.

Responsibility towards human life and safety – lessons from history

Another key aspect of research involving the use of ani-mals is human safety, especially in the context of medical research on humans. The atrocities of medical experiments on humans in Nazi Germany has led the international com-munity to implement strong protection of human subjects and patients. In addition, drug scandals like the thalidomide birth defect crisis in the 1950s and 1960s have led to pro-found changes in drug regulations. The results of this pro-cess have been condensed in the “Declaration of Helsinki”

adopted by the World Medical Association (WMA) in 1964. Importantly, this declaration states that medical research on human subjects is only justified if all other possible sources haven been utilised for gaining information about efficacy and potential adverse effects of any new experimental ther-apy, prevention or treatment. This explicitly includes infor-mation gained from experiments with animals,8 which has additionally been addressed in a dedicated statement by the WMA on animal use in biomedical research.9.

In analogy to the Helsinki Declaration, which has effec-tively altered the ethical landscape of human clinical re-search, members of the international research community have adopted the Basel Declaration to acknowledge their re-sponsibility towards research animals by further advancing the implementation of ethical principles whenever animals are being used in research.10 Further goals of this initiative are to foster trust, transparency and communication on ani-mal research.

Fostering an evidence-based public debate on the ethics of animal research

Transparency and public dialogue is a critical prerequisite for a thoughtful and balanced debate on the ethical implica-tions of using animals in potentially harmful experiments.

However, a meaningful public debate about ethical consid-erations is only worthwhile, if we agree on the facts regard-ing the usefulness of research on animals for scientific and medical progress.

Yet, the contribution of animal models and toxicology testing to scientific and medical progress as well as sub-ject/patient safety is sometimes doubted by animal rights activists. Certainly, in most biomedical research areas, in-cluding those that involve animal experimentation, there is room for improvement, e.g. on aspects of reproducibility or translation of results from bench to bedside. However, there is widespread agreement among researchers and med-ical professionals, together with a large body of published evidence, on the principal usefulness of animal models in general. As for all science, constant improvement of mod-els and careful consideration of whether any model used is still state of the scientific art at any given point of time is crucial for scientific advancement. Also the responsibility to avoid animal suffering as much as possible dictates that new scientific methods and models free of animal suffering are developed with both vigour and rigour.

A fruitful debate needs to be based on these insights and evidence-based common ground needs to be established when discussing ethical considerations and stimulating new ideas. Finally, we need to acknowledge that we are always in the middle of a continuing thought process, in which we very democratically and carefully need to negotiate the importance of different views, values and arguments.

Read more:

[1] Johnson, E. M. Charles Darwin and the Vivisection Outrage. The Primate Diaries (2011).

[2] Feller, D. Dog fight: Darwin as animal advocate in the anti-vivisection controversy of 1875. Stud. Hist. Philos. Sci. Part C Stud. Hist. Philos. Biol. Biomed. Sci. 40, 265-271 (2009).

[3] Darwin, C. R. 1881. Mr. Darwin on Vivisection.

The Times. (18 April): 10. (1881). Available

at: http://darwin-online.org.uk/content/frameset?pageseq= 1&itemID=F1352&viewtype=text. (Accessed: 25th October 2017)

[4] Bentham, J. An Introduction to the Principles of Morals and Legislation. (W. Pickering, 1823).

[5] DIRECTIVE 2010/63/EU OF THE EUROPEAN PARLIA-MENT AND OF THE COUNCIL on the protection of animals used for scientific purposes. 2010/63/EU, (2010).

[6] Russell, W. M. S. & Burch, R. L. The principles of humane experimental technique. (Methuen, 1959).

[7] Guidelines for Researchers. ICLAS Available at: http://iclas.

org/guidelines-for-researchers. (Accessed: 29th November 2018)

[8] WMA – The World Medical Association-WMA Declaration of Helsinki – Ethical Principles for Medical Research Involving Human Subjects. Available at: https://www. wma.net/policies-post/wma-declaration-of-helsinki-ethical-principles-for-medical-research-involving-human-subjects/. (Accessed: 29th November 2018)
[9] WMA – The World Medical Association-WMA State-ment on Animal Use in Biomedical Research. Avail-able at: https://www.wma.net/policies-post/wma-statement-on-animal-use-in-biomedical-research/. (Accessed: 29th November 2018)

[10] Basel Declaration | Basel Declaration. Available at: https://www.basel-declaration.org/. (Accessed: 30th November 2018)