13. Gene-technological Modification of Complex Species
In this chapter I want to briefly outline the technological innovations, that allow interventions into the human genome. For technical details, we may refer to the bio-technical specialist literature. Here, we only want to outline the principles and their application for genetic manipulations.
The entero-bacterium Escherichia coli is the favourite pet of the bio-technologist. Under ideal conditions, E. coli divides every 20 minutes. Through introduction of ring-shaped DNA (plasmids) into E. coli, these plasmids and the genes they carry can be amplified. So, if the bio-technician needs a certain gene or a certain strand of DNA, he can integrate it into E. coli and amplify the plasmid-containing E. coli bacteria in an overnight-culture.
Complex, multicellular organisms do not reproduce clonal, but sexual. Clonal reproduction implies that the complete genome (only modified by mutations) is passed on from one generation to the next. Sexual reproduction implies that the genome of the following generation consists of half the mother’s and half the father’s genome. Nevertheless, clonal growth and multiplication is omnipresent in all sexual reproducing organisms. Mitotic cell separation, the doubling of body cells, is a form of clonal growth and underlies all growth- and regeneration processes inside these organisms. During mitosis one parental cell leads to two daughter cells with identical genomes. Only the formation of germ-cells (sperms, ovules) through meiosis is not clonal, thus making sure that offspring of sexual reproducing organisms have a mix of both parents’ genomes.
Currently a lot is being written about the CRISPR/Cas9-method, which makes targeted genome-modification possible. Indeed, CRISPR/Cas9 enables targeted modification of DNA- and RNA strands in genomes with a previously unknown precission. However, this method is not the first known tool for cutting DNA. Before looking at the CRISPR/Cas9-method, we will first look at the gene-scissors that molecular-biologists have in their tool box for decennies. Restriction enzymes (restriction endonucleases) can targetedly cut DNA strands at certain sequences. As a medical doctoral student, I used circular shaped DNA (plasmids) for amplyfing certain DNA strands inside E. coli bacteria. The gene-strand to be amplified is being inserted into the plasmid, the plasmid is entered into E. coli bacteria and the bacteria are plated out on an agar-plate. To make sure that only bacteria that contain the plasmid grow, an antibiotic-resistance gene is part of the plasmid and the agar plate contains the corresponding antibiotic. Some of the growing cultures consist of E. coli that contain a plasmid, but a plasmid without the insert. A gene for the production of a blue color marker is additionally build into the plasmid, exactly, where the insertion site of the plasmid is. If an insertion worked out and the plasmid contains our gene-strand to be amplified, they interrupt the continuity of the blue color gene. On the next day, one rejects the blue colonies and picks the colonies that are white for further growth in a fluid broth medium.
Cutting DNA strands is a prerequesit for inserting DNA into a plasmid. For cutting DNA, restriction enzymes are being used. The restriction enzyme EcoRI, for example cuts at the recognition sequence 5’-GAATTC-3’ with 3’-CTTAAG-5’ in the antisense-strand. Figure 1 shows the cut schematically:
The CRISPR/Cas9-method builds on long-years of experience with enzyme tools. What makes the method so powerful is its ability to implement targeted changes of the genome of complex living organisms, including humans. In the CRISPR/Cas9-system the target finding and the cutting is done by two different enzymes. With CRISP the target DNA sequence is found thanks to a “guide RNA”, Cas9 is an endonuclease that cuts the corresponding sequences. CRISPR/Cas-system is originally a component of the bacterial immune defence against viruses, with Cas9 being the “destroyer protein”. The RNA sequences contained in CRISPR is equivalent to viral sequences and guide the Cas-destroyerprotein to its target. The biotechnologist synthesizes a “guide RNA”, whose sequence corresponds to the target sequence of the DNA piece to be cut out. The guide RNA anneals to the inverse DNA-sequence of the antisense strand and Cas9 cuts out the sequence. Now the strandends can be reconnected or an insert can be inserted. The CRISPR/Cas-system works in living cells und thanks to the guide RNA, the intervention takes place in different places, where the corresponding target sequence is located. The system has all properties required for gene-therapeutic instruments: Target-precission, usability in living cells and the ability to seek the target in multiple locations.
Regarding the implications of such an intervention, the distinction between somatic gene modification and a gene modification affecting the germline, is important. Somatic interventions are on body cells. A future vision for treating diabetes, for example is the biotechnological production of insuline producing cells. Such an intervention does only affect the treated individual. A germline intervention, in contrast, means modification of germ-cells (sperms, ovules). Half of the genome of the sperm and half of the genome of the ovule mix and form the genome, from which the embryo emerges through mitotic cell dividsions and finally a new human being, who then carries the modification introduced into germcells. Germcell modifications are therefore passed on over the generations, somatic cell modifications are not passed on. In principle germline interventions could be used for preventing (repairing) congenital diseases. At the moment they are regarded to be too risk ridden (90).
With CRISPR/Cas9, a genetic tool is available that could be compared with the search-replace function in word-processing-programmes, only that the information is not semantic-verbal, but in form of genetic traits written in the language that we recognize as the genetic code.
Preceeding the “Second International Summit on Human Genome Editing“ in 2018 in Hongkong, the claim of a Chinese scientists of having introduced a modification of the CCR5-gene of recently born twin sisters, triggered an agitated international debate. Dr He Jankui introduced targeted mutations into 7 embryos prepared for fertility treatment (implementation into a woman, who wants to get pregnant this way). In all cases the father was HIV positive and the mother HIV negative. One pregnancy emerged (91). It is known that a mutation oft he CCR5-gene protects from HIV. However, an infection of the ovula through HIV via a fertilizing sperm is very unlikely an becomes even more unlikely if the sperm undergoes several washing procedures before in vitro fertilization. The potential benefit of the genome treatment, could only be seen in a protection from HIV-infection in later life. However, this protection could have been traded off against a higher vulnerability for other diseases. It is known that individuals with CCR5-mutations are prone to have more severe courses for west nile fever virus infections. Furthermore, gene editing procedures are still in an experimental stage and can lead to mutations in other sites (“off Target”), that in turn can result in inprevisible genetic problems such as increasing the cancer risk. There is even a claim that the CCR5 mutation could act in a way that shortens the life-span, however the claim of CCR5 shortening life expectancy could not be statistically substantiated and was retracted (92). The CCR5-gene codes for a surface receptor that is being used by HI-viruses as a co-receptor for the infection of lymphnodes and macrophages. This is why individuals with a modified (defect) CCR5 receptor are protected from HIV. What makes this intervention particularly controversial is the possible association of CCR5 with brain functions and cognitive abilities. Mice with a CCR5 deletion had a better memory (93). In mice it could also be demonstrated that blocking CCR5 leads to a better regeneration of neurons after a stroke. This is consistent with the observation that humans with a CCR5 delta32 mutation (that protects them from HIV) show a faster neuroregeneration after the stroke (94). Individuals with a CCR5 delta32 mutation seem also to reach longer schooling times (and thus higher education levels). All in all, it seems plausible that the genetic intervention could have an effect on the cognitive functions of the twin sisters, probably in the sense of an improvement (95).
In February 1997, Dolly became the most famous sheep between North- and South pole and entered into our collective memory. Dolly was a cloned sheep. She was cloned in Edinburgh from a fully developed somatic cell (body cell, not a sperm or ovula-cell) and thus became the first cloned mammal. For the cloning process, a genome containing cell nucleus from udder cells of “Fin Dorsett“ sheep were injected into the ovula-cells of “Scottish Blackface“ sheep. From the 277 prepared ovula-cells, 29 embryos emerged, from which one embryo survived. Dolly was carried out by a surrogate mother sheep of the species “Scottish Blackface“. In an interview with the New York Times, Ian Wilmut (head of the laboratory) reported that all previously born cloned animals died shortly after birth. Apparently, Dolly was not the first cloned mammal that was born, but the first cloned mammal that survived.
Dolly reached an age of 6 years. Usually, the life expectancy of sheep is around 10 years, 20 years at most. Dolly was euthanized, after having contracted sheep-retrovirus and developed lung-adenomatosis, a broncho-alveolary cancer. The circumstances around her death appear independent from her procreation by cloning. However, Dolly had strong arthrosis and arthritis, this is why there was a debate if she may have aged earlier, as the implanted cell-nucleus were taken from an adult animal with correspondingly “aged genome” (96).
Risk assessment for the individual, the person, whose genome is modified is rather speculative. It is simply not possible to predict without data from many such modifications. Also, if a risk has manifested, for example a disease, it will be very difficult to tell, if this is a consequence of the genome modification, especially if a long time lies between the two events.
Let us start with considering the risk of cloning, actually a biotechnological procedure, which does not imply any change of the genome. To the contrary: cloning means to pass on an unchanged genome of an individual to the next generation, thus even switching off the natural mixing of the parental genomes. For cloning, the nucleus of an oval-cell is being removed and replaced with the diploid genome (2×23 chromoseme in humans) of a cell-nucleus of the individual to be cloned. In every somatic cell of our body, we have 2×23=46 chromosomes. Sperms and ovules are haploid, thus containing a single set of 23 chromosomes. Sexual reproduction implies that the haploid set of chromosomes of the ovula (1×23) and the haploid set of chromosomes (1×23) of the sperm cells merge and exchange parts of their chromosomes forming a new diploid set of chromosomes. In somatic cells of our body, we have diploid sets of chromosomes.
The clone sheep Dolly was created using a diploid set of chromosomes (2×27 chromosomes in sheep) from a somatic cell (udder cell), which was injected into the denucleised ovula cell. Her genome did not newly emerge by fusion of two sets of chromososmes, but simply consisted of an already existing diploid genome. The injection of a diploid genome from a somatic cell into a denucleised ovula cell sounds mechanically simple, however one has to keep in mind that for creating Dolly, 277 ovula had been injected, 29 embryos emerged, but only one embryo survived – Dolly.
Something one could forget ist hat Dolly started her life, when she was born, but that her genome comes from an organism, that was already 6 years old (97). Was Dolly’s genetic age 6 years older than her age? Does cloning over several generations lead to cumulative damage through overaged genomes? The resarchers abstained from further propagative cloning of Dolly. Indeed, the telomere regions of Dolly’s chromosomes were a bit shorter. Telomeres are terminal regions of chromosomal DNA. The play a role for decoiling DNA strands for the copying of the genome for cell division. At the end of the terminal regions, short parts of DNA are not being copied, so that the daughter strains are always slightly shorter than the template strand. Therefore, the length of telomeres decreases over cell generations from cell division to cell division.
The research group that created Dolly, created more cloned sheep using the same udder-cell genomes. According to the researchers, these sheep (once they survived the postnatal period) lived a normal, healthy life without signs of premature aging. A cohort of 13 cloned sheep, who were examined at the age of 7-9 years, had age appropriate joints, blood pressure and metabolic factors (98). Another question circles around the ability of cloned animals to produce offspring. Dolly gave births to 6 healthy lambs, who were all fathered the natural way by the same ram, David (99).
Procedures that carry a lot of problems and failures in the initial phase can become routine procedures, whose results can improve with routine and experience. Meanwhile, animals are not only being cloned in research laboratories, but already for commercial purpose. To meet the increasing demand for pork in China, industrial breeding of cloned pigs has been established (100). Scientific research on cloning has suffered a bit under the scandal around the South Corean scientist Hwang Woo-suk, who apparently forged many of his spectacular stemcell and cloning results and projects. The dog Snuupy, the first cloned dog in 2005, is regarded to be real. In contrast to cloning cloven-hoofed animals, the cloning of dogs is more difficult, due to the limited phases, during which bitches produce ovula-cells that are fertile.
For the society, or even mankind, somatic gene-modifications that are not passed on to the next generations, are less consequential and risky than germline modifications (that are passed on). Somatic genome modifications have no direct effect on the coming generations. The potentially resulting injustices that can emerge, if genetic self-optimization becomes a question of being rich or poor, will be discussed in the chapter “Access to the resource genetic optimization”. If we factor out the potential injustices, it seems difficult to principally reject self-optimization wishes. In case of a somatic genome-modification, the medical risk suffering damages and side effects is carried exclusively by the person, whose genome gets modified. The person, who gives an informed consent to the procedure is solely affected in contrast to germline interventions that affect the offspring, who have no possibility to take part in the decision. (If we apply this line of arguments consequently, however, even the generation of a child and human life as such would be a problem, as no human being on earth took part in the decision to be thrown into life; – the antinatalists take such a radical position)
At the moment we have no ideas about the risks of somatic genome modifications for the wellbeing of humans, whose genome gets modified. To gather such knowledge cell cultures and animal models will reach their limits and empirical data from humans would be required. There surely would be a transition phase, in which damaging effects would not be known yet and certainly not controlled, but learning about such damages and controlling them will not be possible without experience from humans and their sacrifice. The closest situation for getting ethical permission is the repair of severe genetic defects justifying such an experimental cure. For genetic “lifestyle” interventions with enhancement character, one will probably count on the bravery of volunteers.
Especially diseases with a single gene defect, or with failure of the function of a single gene are interesting candidates for somatic gene-therapeutic approaches. Congenital retinal dystrophias are a heterogenous group of congenital retina degeneration that leads to increasing debility of sight and ultimately blindness. More than 100 gene-loci have been identified, where mutations cause this severe eye disease. Although, there are so many gene-loci for this disease, it is an attractive target for somatic gene-therapy, as the disease in the individual is caused by only one mutation (and not by interplay of multiple mutations).
Leber congenital amaurosis occurs in Briard dogs and in humans and in both species is caused by a mutation of the so called RPE65 gene. The availability of a good animal model was an important prerequisite for establishing a somatic gene therapy. Furthermore, the nature of the disease, allows for gene-therapeutic injections in targeted locations (the affected retina-cells). Adenoviruses are used as vehicles for the gene strand that contains normal copies of the RPE65 gene. The targeted injection into the retinal tissue minimizes systemic side effects (101). At the end of 2017, this gene-therapy was approved by the FDA (American drug regulatory agency) (102). Nevertheless there remain systemic risks, and there have already been cases of death cause by gene-therapeutic adenovirus infections (103).
So, in principle it is possible, to cure monogenetic diseases and by now there are even officially approved therapies.
Nevertheless, the number of people with congenital genetic diseases will rather be decreased by prenatal diagnostics (and abortion) than by therapy. At the moment prenatal diagnostics still implies amniocentesis, which means sticking a hollow needle into the amniotic sac to win foetal cells, whose genome can then be examined. Foetal cells can also be found in the blood of the mother, though in very little concentrations (< 1:1 million cells). Nevertheless, prenatal diagnostics from foetal cells won from maternal blood, seems to become a realistic possibility (104). In parallel more and more genetic loci can be screened in one examination. If prenatal diagnostics becomes methodotologically easier, less invasive and more-encompassing, more and more gene defects can be detected early. The corresponding pregnancies are often terminated by abortion.
Prenatal diagnostics brings about many ethical questions, for example, which gene-loci should be screened. The disaeses linked to a genetic defect have to be severe enough to justify an abortion. Unfortunately, it is not always possible to clearly predict a phenotype from a genotype. How to deal with genetic defects, that do not cause any harm in some individuals and severe diseases and debilitating disabilities in others? Furthermore, prenatal diagnostics take place in a relatively late state of pregnancy, so that abortion implies the killing of a relatively far developed life.
Aneuplodia screening (examinations for chromosomal anomalies) of foetal cells from maternal blood has already been done, however the cells were gained in the 23rd week of pregnancy (105). With good medical care, premature born babies can already survive at this age. Aneuplodias imply that a chromosome is missing or one additional chromosome can be found. Most aneuplodias lead to spontaneous abortions, however humans with Trisomias 21 (Down syndrome) can live a long and happly life.