What is the process of inserting normal genes into human cells to correct genetic disorders?

5.2 Germ Line Gene Therapy

Germ line gene therapy is much more controversial (Nelson 2000). It would introduce ‘normal’ human genes into the eggs or sperm of parents, or into the fertilized egg or early embryo of the offspring. The goal would be to change the eventual child's genetic inheritance. This could be done in order to avoid a genetic disease or in order to introduce an ‘enhancing’ genetic variation. There have been no trials of human germ line gene therapy; indeed, there is an informal moratorium in the scientific community on trying such experiments in humans. Both its feasibility and its value are unclear.

New genes have been successfully introduced into the germ lines of other mammals, but with very low efficiency. At the same time, pre-implantation genetic diagnosis allows parents to choose embryos based on their genetic variations, as long as the parents themselves produced the desired variations. If not, donated eggs or sperm would be a much safer and easier way to introduce the desired genes than somatic cell gene therapy. Germ line gene therapy may turn out to be most important as a barrier to somatic cell gene therapy. If germ line gene therapy were banned, researchers using somatic gene therapy might need to make the difficult showing that the transplanted genes could not ‘infect’ the patient's germ cells and thus constitute inadvertent germ line gene therapy.

2.2.2 Germline gene therapy

In germline gene therapy, DNA is inserted into the reproductive cells (eggs or sperm) in the human body. Germline gene therapy will correct the genetic variants of the reproductive cells of an individual, and this would be passed down to future generations. This therapy removes a hereditary disorder from a family line forever. Hereditary disorders occur at human’s are possibly inherited from the germline cells. However, curing these diseases is possible only through modifying their nuclear and mitochondrial DNA mutations in preimplantation embryos, which is commonly known as germline gene therapy (Wolf, Mitalipov, & Mitalipov, 2019).

INTRODUCTION TO GENE THERAPY

Germline gene therapy is the replacement of genes in which an offspring would inherit a new trait. This type of gene modification is still in the early stages of human medical intervention but examples do exist, such as in cows modified to have elevated levels of milk production or the capability to secrete human hormones, or “knockin” and “knockout” mouse models, used for decades to elucidate gene function. The second, currently more medically viable and potentially less controversial, is somatic gene therapy. Somatic gene therapy is the introduction of genetic material directly into the body after development. There are three categories of somatic gene transfer: in vivo, in situ, and ex vivo. In vivo is the introduction of genes directly to the body via the blood stream; for example, in situ involves targeting a specific organ for gene transfer, such as the eye. By contrast, in ex vivo gene transfer a particular class of cells are removed from a patient, modified, and then replaced back into the patient in order to treat an abnormality or regain a particular function.

The notion of gene therapy arose in the 1960s, and with the advent of stable cell lines and advancing techniques of DNA isolation and manipulation, that idea became more of a reality. Early techniques involved primarily the use of bacteria, such as Escherichia coli, and utilized the fact that their genomes are comprised of circular DNA which can be shared with other bacteria in a process known as conjugation. This type of gene transfer is what gives bacteria the ability to share resistance genes among colonies. Microbial molecular biology soon gave rise to advances in techniques such as gene cloning and circular plasmid DNA construction. These techniques offered insights into microbial genetics and in addition led scientists to ponder whether these concepts could be applied to human maladies.

One of the first human diseases to be investigated for gene therapy was the hypoxanthine guanine phosphoribosyl transferase (HPRT) deficiency associated with Lesch–Nyhan syndrome. Researchers studying this disease were able to demonstrate that exogenous DNA was capable of uptake and expression into mammalian cells. Unfortunately, the methods available at the time were unable to produce stable expression efficiently in these cells.1 It was not until the late 1960s through early 1970s that more viable methods came into development. A major discovery arose when researchers were able to elucidate key events in the ability of polyomavirus to transform cells, integrate, and stably express their DNA.2–4 This, in addition to chemical transformation methods (i.e., calcium phosphate), enhanced researchers’ ability to insert genes into cells.5 It was at this point that the proof of principle was illustrated with stable introduction of the bacterial HPRT gene analog into deficient mammalian cells.6 It was also during this period that it was postulated, based on the insight gained regarding polyomaviruses, that it may be possible to manipulate other viruses; amid a genetic revolution this gave way a decade later to the development of retroviral vectors and then to several other viral-mediated gene transfer techniques, several of which are used today and are currently the mainstay of gene therapy applications.

10.10 Genetic Therapies of Mitochondrial Diseases

Both somatic and germline therapies are now being developed for mitochondrial disease. Gene therapy treatment of somatic tissues involves both the direct complementation of nDNA mitochondrial gene mutations and various efforts to complement pathogenic mtDNA mutations. In a mouse model of Ant1 deficiency, the nDNA null mutation was successfully complemented in muscle by injection of an adeno-associated virus (AAV)-borne Ant1 cDNA [292]. This approach will likely be used to treat various mitochondrial myopathies and cardiomyopathies in the years ahead.

Efforts to introduce DNA into the mitochondrion have been reported using both DNA–protein aggregates [293] and by targeting AAV to mitochondrial membranes [294]. The most actively pursued approach for genetically treating mitochondrial disease has been the allotopic expression of mtDNA genes. In this procedure, mtDNA genes are cloned and the mtDNA genetic code adjusted to be optimal for the nucleus–cytosol compartment. The mtDNA-derived cDNA is then linked to an N-terminal mitochondrial targeting peptide, nuclear gene expression elements added, and the construct introduced into the nucleus via AAV. Expression of the transgene results in the cytosolic synthesis of the mitochondrial polypeptide which is then imported into the mitochondrion by the N-terminal targeting peptide. When the polypeptide is processed, the hope is that it will be incorporated into the appropriate mitochondrial enzyme complex. This approach is being actively pursed for treating the common LHON ND4 nt 11778A>G (R340H) mutation by injecting the virus into the eye vitreous to transduce the affected retinal ganglion cells [295–304]. A newer approach could be to have the nucleus express allotopic mRNA that is imported into the mitochondrion. Inside the mitochondrion, the imported mRNA could be translated on mitochondrial ribosomes and directly introduced into the appropriate complex [305–307]. This approach could have the advantage that the mRNA with the mitochondrial genetic code could not be translated in the cytosol, thus avoiding the accumulation of toxic cytosolic protein aggregates.

Most recently, mtDNA mutations have been eliminated thrugh germline mitochondrial replacement therapy. Two procedures are being developed: zygote pronuclear transfer (PNT) and oocyte spindle transfer (ST). In PNT procedure the oocyte of the affected woman is fertilized and the two pronuclei are transferred via a micropipette into an enucleated recipient zygote from a woman with normal mtDNAs [308,309]. In the ST procedure, the spindle from the metaphase II oocyte is transferred into an enucleated oocyte with normal mtDNAs followed by fertilization and implantation [310–314]. The birth of one spindle transfer “three-parent baby” has been reported in the literature. This child was born to a mother who had lost two children to Leigh syndrome due to high levels of the mtDNA ATP6 8993T>G (L156R) mutation. One implanted ST blastocyst gave rise to an apparently normal boy carrying between 2% and 9% 8993G mutant mtDNA in his various tissues [315,316].

Human Dignity

A related objection to germ-line gene therapy is that it offends against human dignity, an idea derived from Kant, specifically his categorical imperative that we should always treat people as ends in themselves and not merely as means to an end. Yet it is hard to see how seeking to cure people, or more accurately, prevent them from developing disease, is treating them as means to an end. What end is it that we are treating them as a means to? Perhaps it could be claimed that the end is one of a healthy society, and using the therapy is treating people as merely a means to this end. But then surely this is true of all medical treatment. Rather, medicine is administered to treat individuals, to make individuals better – a side effect is the improved health of society as a whole. Proponents of the human dignity argument have to show why trying to prevent someone getting a disease is not treating them as an end in themselves.

Of course, such a case can be made for experimental developments of gene therapy, where the patients are, to some extent, human guinea pigs. In this case, it could be claimed that use of (experimental) germ-line gene therapy treats people as means to an end (that end being experimental results) rather than ends in themselves and could thus be seen to act against human dignity. Perhaps such arguments could be used against the development of germ-line therapy; even if the use of such therapy is ethical, its development is not. The trouble with this position is again the similarity to standard medical treatments. Were these considered as acting against human dignity when they went through experimental testing?

Gene Therapy Ethics and Regulation

There are major differences in the ethical issues pertaining to somatic as opposed to germline gene therapy. In most countries, germline gene therapy, because of its potential effect on future generations, is appropriately outlawed. Our limited understanding of the complex interactions that have shaped human evolution, together with societal and cultural considerations, precludes the possibility of conceiving of responsible programs for germline genetic modifications in humans, as well as germline gene therapy approaches. However, somatic gene therapy is encouraged and performed worldwide under strict regulatory authority with remarkable congruency of guidelines in different countries and global constituencies. The concept that gene therapies constitute novel biologic drugs provides an appropriate framework for regulatory oversight.

4.2.2 Gene-therapy

It is hoped that the incorporation of human or nonhuman genes in body cells (somatic gene-therapy) or the human germline (germline gene-therapy) will be a major contribution to the therapy of diseases with a genetic component—hereditary as well as non-hereditary. Somatic gene therapy is seen as a relatively unproblematic method from the ethical (not medico-technical) point of view, since the transferred genes are not inherited by the descendent of the treated individual.

In stark contrast to this, germline gene-therapy is controversially debated. Since the transferred genes to germline cells are inherited by all offspring, the question arises whether such an intervention in the genetic constitution of an unborn individual can be legitimated and, on a more general level, whether germline therapy leads to a new form of eugenics or genetic enhancement. Since most authors hold that if germline gene-therapy were to be applied, it could not be exclusively confined in the long run to medical application, the debate therefore centers on the question whether the intervention into the lives of future persons by genetic enhancement is different in any morally relevant way from classic interventions such as education (Agar 1998)

Whose Harms, Whose Benefits?

The same question can also be extended to stem cell research and gene treatments. Approximately 20 years ago, the promise was that somatic cell therapies and germ line gene therapies would start to make an impact in 5–10 years’ time, but very few useful applications have been invented so far. The champions of stem cell research now present the same arguments, but there is no guarantee that any actual benefits will be seen in the near future. In the meantime, vaccination programs continue to be postponed, and drugs for fatal illnesses are not sold in developing countries at reasonable prices. A handful of people in the affluent West enjoy the fruits of high technology, whereas the majority of humankind is confronted by the more mundane evils of famine, water shortages, and infectious diseases.

In addition to not benefiting a considerable portion of humanity, genetic advances can actually harm a number of people or lead to their exploitation. Individualized stem cell therapies are a case in point. The idea is that when a person suffers from the malfunction of an organ or the lack of vital tissue, then a somatic cell is taken from that person’s body and cloned for the production of the required organ or tissue. For each treatment like this, at least one human egg is needed because the ova of other species cannot currently be adequately used. However, these eggs need to come from somewhere or, more precisely, from somebody. How will this be accomplished? If women are expected to donate all the required ova, will they be willing to do so? If some women are expected to sell their eggs, what social and political consequences will this have? Legislators already struggle with the implications of organ sales and will not be likely to soon derive universally approved solutions to the issues surrounding the commercialization of the human body.

Harm can also be caused by germ line gene therapies, which remove the genetic mutation from the persons treated and from their descendants. Even with the completion of the Human Genome Project, our knowledge of the interaction of genes is extremely limited, and we cannot determine what effects the removal of a particular element in our constitution would have on the whole system. It is possible that in their zeal to eliminate the causes of ailments, genetic engineers may inadvertently do away with something truly vital in the process.

In light of these considerations, it is evident that instead of making people healthier and happier, gene technologies could make them sicker and more miserable. Also, even where they can be moderately successful, the resources consumed on their development could well be better spent in more down-to-earth social and public health projects.

5 Gene Therapy

The goal of gene therapy is to provide a functional protein to an individual whose genotype leads to a disease because the protein is missing or modified so that it is not functional. There are two potential modes. In germ-line therapy, the modification would be heritable, and the individual's offspring potentially would not be affected. In somatic cell therapy, only the treated individual would have the modification. There have been many arguments against germ-line therapy including our inability to know whether potentially harmful changes in the sequence have been made inadvertantly. All of the trials to date have been in somatic cells for the treatment of inherited disease and cancer by control of cell death.

Two strategies are being attempted. For those disorders for which bone marrow transplantation has been effective, the gene is introduced into marrow cells, the population of which is then expanded and infused into the patient. The techniques for gene introduction are similar to the ones previously discusssed: viral vectors and chemical or physical techniques. An immune deficiency disorder, adenosine deaminase deficiency, was the first attempt reported. Although the modified cells express the enzyme, repeated infusions of treated cells have been necessary, and the patients have continued to receive chemically modified enzyme, so it is not clear whether or not gene therapy alone is sufficient.

Other trials have used viral vectors to treat target organs, such as the lung in cystic fibrosis. The therapeutic trail of an adenovirus-mediated transfer of a gene to the liver has received general media attention because of the death of a participant. Evidently, other participants in gene therapy trials have died, although it is not clear how many of those deaths are a result of the procedure (Strachan and Read 1996).

Anticipated Returns on Educational Investment

This book will allow the student to comprehend the language, the direction, and the uncertainties in the field of genetics and genomics. It will help distinguish between such critical words as “cause” and “susceptibility,” and what is meant by “necessary and sufficient.” It will remind students of those instances when geneticists halted their own work on recombinant DNA technology, germ line gene therapy, reproductive cloning, and more, because they were concerned that the benefits in the work were exceeded by the risks. It will allow students to interpret and criticize what they encounter in the media and, thereby, be able to develop an informed position on a wide range of topics that will affect their lives.

REVIEW QUESTIONS AND EXERCISES

Discuss briefly four reasons why the field of human genetics has been at the forefront of science and medicine during the past half century.

Depending on one’s point of view, the field of human genetics either raises hopes or provokes fears. Discuss briefly three kinds of hopes and three kinds of fears.

As each new genetic technology has been developed, it has run into intense opposition from a segment of society. Describe briefly three such technologies and the origin of the opposition engendered.

An article in a major US newspaper was entitled “Gene Screen: Will We Vote Against a Candidate’s DNA?”

Is this headline mere attention grabbing? Why, or why not?

What societal changes would have to occur before mandating disclosure of a candidate’s genome sequence would be appropriate—if ever?