If one were to demand today that all medicines that have come into contact with genetic engineering – in whatever form – in the course of their development would have to be withdrawn from the market, the supply of patients would be massively endangered. This applies not only to biologicals, but also to newer small-molecule drugs , all of which are characterized preclinically in model systems developed using genetic engineering methods. If all genetic engineering work were generally banned, a large proportion of "life scientists" would probably be able to hang up their nails. In the meantime, active substances for gene therapy have also been approved in Germany, which, together with other so-called Advanced Therapy Medicinal Products (ATMPs), enrich the therapy options for severe illnesses.
The universal genetic code as a basis
If you look at the history of genetic engineering, you usually go back to the 19. Go back to Gregor Mendel (1822 to 1884) and his rules of heredity in the nineteenth century. However, very important milestones were the identification of the DNA as a carrier of the genetic information by Oswald T. Avery in 1944 and the elucidation of the DNA double helix by James D. Watson and Francis Crick 1953.
After experiments by Erwin Chargaff made it clear how the DNA bases thymine and adenine and cytosine and guanine assemble, and Matthew Meselson, together with Franklin W. Steel identified the semiconservative replication of DNA, all the essentials for programming all living things had been established. The only thing missing was a solution to the question of how the information from the genome is actually converted into a protein. In 1961, Marshall Nirenberg and Heinrich Matthaei proved Francis Crick’s thesis that a codon consisting of three bases each carries the information for an amino acid.
Figure 1: Basis of DNA recombination. After hydrolysis with a restriction endonuclease, matching ends are formed that can be re-ligated with each other. / Photo: I. Zundorf
So it was only a matter of time before, as a result of a joint effort by many researchers, the genetic code was decoded, which, interestingly enough, applies equally to all living organisms. The genetic information for insulin from humans, for example, is understood in bacteria just as it is in the fruit fly or in the tobacco plant.
In parallel to the experiments on DNA, research on bacteria, especially the intestinal bacterium Escherichia coli, and bacteriophages was carried out; several enzymes were isolated and characterized from prokaryotic cells. In 1956, Arthur Kornberg identified for the first time a DNA-dependent DNA polymerase that was able to synthesize a DNA strand in a test tube using a template, and which was later named after him (DNA polymerase I or Kornberg polymerase).
Figure 2: Principle of genetic engineering: once an interesting gene has been identified from a source organism, for example the insulin gene from humans, it can be isolated, combined with new control units and introduced into any target organism. This target organism is thus genetically modified, i.e. a GMO, and capable of producing human insulin. / Photo: I.Zundorf
The next step was the discovery that bacteria defend themselves against DNA introduced by phages by means of so-called restriction enzymes. First, Werner Arber and Matthew Meselson identified type I restriction endonucleases in the 1960s, which recognize a DNA at a specific site but then randomly make a cut in the double strand some distance away from it. Type II restriction enzymes proved to be much more helpful for molecular biologists. This type of endonuclease recognizes and cuts a very specific, four- to eight-nucleotide-long sequence of bases within DNA , which has double symmetry. If different DNA molecules are cut with the same enzyme, the same ends are produced in each case, which can then be reconnected like matching connectors (Figure 1). The enzyme required for this, a so-called DNA ligase, had been developed by Bernard Weiss and Charles C. Richardson already described in 1967.
All the conditions were now in place for recombining DNA to produce genetically modified organisms (GMOs) (Figure 2) – an experiment that Herbert Boyer and Stanley Cohen, together with Annie Chang and Robert Helling, published in 1973, just 20 years after the DNA structure was elucidated. At that time, with the meager technical possibilities, this was dramatically fast.
Key experiment with huge potential
Already at that time, some recognized the potential, but also the dangers, of this key experiment. Nobel laureate Joshua Lederberg predicted that "the concept of the pharmaceutical industry could change completely with the production of biological agents such as insulin and antibiotics" – which was indeed the case within a short period of time.
With an eye on the profits that could beckon from this development, Stanford University , where Stanley Cohen worked, quickly initiated patenting of the process. Over the next 17 years, royalties on the exploitation of the method generated a total of $255 million, which went to Stanford University and the University of California, where Herbert Boyer worked. Boyer also had the creative idea of founding Genentech, the first biotechnology company, in April 1976.
As early as 6. September 1978, only two years later, the press release appeared that human insulin had been successfully produced in bacteria. After that, it took another four years until the first genetically engineered insulin from Lilly was approved in 1982.
Insulin therapy without genetic engineering- this is no longer imaginable today. / Photo: AdobeStock/6okean
In the meantime, there are 220 (as of: 27. February 2019) genetically engineered compounds approved in 270 drugs. These 220 active ingredients are not all expressed in genetically modified bacteria. The expression of human interferon in the baker’s yeast Saccharomyces cerevisiae was described as early as 1981, and Saizen®, a somatropin produced in a murine C127 cell line, was launched in 1989.
Thus, a whole set of different expression systems from bacteria to higher eukaryotic cells is available. In addition to various culture cells, transgenic animals (rabbit for conestat alfa and goat for antithrombin III) were and still are used for the expression of recombinant proteins. The fact that all this is possible is due to the universal genetic code, which is understood throughout living nature.
Discussions about genetic engineering
The first genetically modified animal for which a patent was granted was the cancer mouse in 1988. A functional copy of the human activated oncogene vHRas was inserted into the genome of this mouse. This case gave rise to discussions about the extent to which animals – even if they have been equipped with new genes – can be patented at all.
As early as the 1970s, there were warning voices that saw dangers in the new technology. Responding to a worldwide call, international scientists directed by Paul Berg , a pioneer of genetic engineering , gathered at the Asilomar Beach Conference Center in Pacific Grove ( California ) in 1975 to develop safety concepts and guidelines for the production and handling of genetically modified organisms. After the U.S. Food and Drug Administration published the "Guidelines for Research on Recombinant DNA" in 1976, the newly established Central Commission for Biological Safety (ZKBS) in Germany also drew up corresponding "Guidelines for Protection against the Hazards of In Vitro Recombinant Nucleic Acids" in 1978. These were binding for all government research institutions and publicly funded projects.
Only after the Council of the European Economic Community issued two directives in 1990 on the contained use of genetically modified microorganisms and on the deliberate release of genetically modified organisms into the environment, did the German Bundestag also pass the first "Genetic Engineering Act". The guiding principle was to protect humans and the environment from the harmful effects of genetic engineering. In addition, a legal framework for the research, development, use and promotion of genetic engineering should be established.
The definition of a genetically modified organism contained therein states that it is an "organism, other than a human being, whose genetic material has been altered in a way that does not occur under natural conditions by crossing or natural recombination" (GenTG § 3 Abs. 3). However, this did not mean that genetic engineering methods could not be applied to humans – the possibilities of repairing a genetic defect with an intact copy of the gene, for example, were too tempting.
Incidentally, the cloned sheep Dolly, which was presented to the public in 1997 and also caused a great deal of excitement, was not a genetically modified organism . This animal was created by transferring a nucleus from an adult cell to an egg cell that had previously had the nucleus removed – entirely without recombining DNA.
Gene therapy : initial trials and problems
After the gene for adenosine deaminase was successfully introduced into human hematopoietic cell lines by means of retroviral vectors in 1989 and expressed there (Figure 3), the first gene therapy was attempted in 1990 . Ashanti de Silva, who suffered from a severe combined immunodeficiency due to an inherited adenosine deaminase deficiency, was treated with this revolutionary new form of therapy at the age of four. She received a total of eleven infusions of retrovirally modified own T cells within two years. She showed no serious side effects, was able to lead a halay normal life and is still alive.
Figure 3: Principle of gene therapy: In order to genetically modify body cells of a patient, the newly combined DNA can be introduced into the body by means of liposomes or viruses (in vivo gene therapy, left side). Glybera® and Luxturna® use this principle. Alternatively (right picture), body cells can be removed, genetically modified ex vivo and then reinfused. Strimvelis®, Kymriah® and Yescarta® work like this. / Photo: I. Zundorf
Gene therapy suffered a major setback in 1999 when 18-year-old Jesse Gelsinger died four days after undergoing gene therapy treatment. The young man suffered from a mild form of a hereditary metabolic disorder of the liver and had volunteered for the gene therapy trial. Catastrophic was also a report from France : children who had received gene therapy for X-linked severe combined immunodeficiency (SCID-X1) showed an increased abnormal proliferation of genetically modified T-cells.
At this point, at the latest, the question began to be seriously asked as to why what was actually such a good and simple therapeutic approach to solving genetic problems did not work as everyone had hoped. It was time to leave the patient’s bedside for a while and go back to the lab to do some homework that had been criminally skipped over in the excitement of the process.
How does foreign DNA get into the cell??
One problem is the very inefficient transfer of therapeutic DNA into the target cells . Even in the case of bacteria , some of which already have a "natural interest" in taking up new DNA and thereby increasing their genetic variability, in the best case only 0.1 to 1 percent of the cells used actually take up nucleic acid from the outside. In eukaryotic cells, the percentage is even much lower.
This is not a problem for cells that can be cultivated on a large scale. Bacteria, yeast and CHO cells can be multiplied at will, and suitable selection systems can be used to distinguish the genetically modified cells from the wild-type cells. For effective gene therapy, things look quite different: Here, as many of the patient’s cells as possible should take up the new genetic information and also convert it into protein.
Many different DNA transfer methods, chemical-physical as well as biological, have been developed in recent years to introduce genes into cells as efficiently as possible. Biological systems take advantage of the fact that viruses are naturally capable of inserting their genome into their host cells. It goes without saying that viruses used for gene therapy must be modified in such a way that they are not pathogenic (anymore).
Nevertheless, these particles are foreign bodies for our immune system. In an in vivo gene therapy approach, in which the genetically manipulated viruses are introduced directly into the body, they trigger corresponding defense reactions. This was ultimately the cause of Jesse Gelsinger’s death. He was given the maximum amount of genetically modified adenoviruses in the gene therapy study with 3.8 x 1013 particles. Although the viruses were applied directly via the hepatic artery, they spread throughout the system and led to a massive immune response with multi-organ failure. Here, it was not the gene to be introduced that was the real problem, but rather the foreign structures of the viral surface.
What are suitable gene transfer vectors?
Adenoviruses are still among the most widely used transfer vectors in gene therapy studies. In addition, apathogenic variants of retro-/lentiviruses, adeno-associated viruses as well as vaccinia, herpes and some other viruses are also used. The decision to use one vector or another depends on the amount of genetic information to be introduced into the target cell and whether the new nucleic acid should integrate into the host cell genome or be extrachromosomal (Table 1).
One of the most common vectors in gene therapy: adenovirus / Photo: Adobe Stock/petarg
A major disadvantage of non-integrating vectors is that the genetic information is lost over time and the expression of the introduced gene is only transient. In contrast, vectors integrated into the genome remain in the daughter cells of dividing cells: Gene expression can persist for a long time. However, random integration of the introduced DNA at any point in the host cell’s genome can lead to activation of a proto-oncogene or inactivation of a tumor suppressor gene, and consequently to tumorigenesis. This is exactly what happened to the children after the therapy of congenital SCID in France.
This problem could be solved with the help of the new tool CRISPR/Cas9. In 1987, a research group led by Atsuo Nakata in Japan discovered a collection of repetitive short sequences interrupted by variable regions in the bacterium Escherichia coli. However, it took until 2013 for the work of Jennifer Doudna and Emmanuelle Charpentier to turn this into a very effective tool for a process now known as genome editing, which was dubbed "breakthrough of the year" by the prestigious journal "Science" in 2015. If the intact gene is introduced into the host cell together with a suitable guide RNA and the Cas9 enzyme, the defective gene can be cut in a very targeted manner and replaced by the new copy.