Legal and ethical issues about the ‘new genetics’ have become favorite subjects for general conversation. However, the quick expansion of medical and biological research contrasts with a steadily growing data dearth among the public. For this proof scientists and physicians are called upon to notify political, religious and other circles in such a path as to provide a real basis for the conversation relating to the restrictions which reason and behavior impose upon manipulations of the human genome. Only then will the clauses for a significant bioethical valuation of the existing issues be performed.

Last advance in molecular genetics motions the eventuality of inserting human genes into human cells. This could help to the cure of a few life-threatening or severely disabling congenital diseases and possibly of some other provisions. Gene therapy would then be of real assistance to sick people, if no other forms of treatment were available.

On the other hand the measures employed in gene therapy should be safe and not result in new problems which might impair the physical and mental health of the cured patients or their environment. In this glance, methodological aspects of possible human gene therapy are considered in the light of the possible feasible uses of this new form of medical cure.

In the conference on the possible uses of gene therapy, a recognition must be made with attention to the following two faces:

1) The molecular manner by which the genome of the patient’s cells is improved

2) And whether the cure is to be made on germ-cells or somatic cells or on embryonic cells. Genetic changes in somatic cells are not supplied to the child of the patient.


Methods and ways of gene therapy

The following forms of gene therapy can be crossed:

1) The introduction of a gene at an unknown place in the genome, when a functional genetic factor is missing (gene insertion),

2) Elective substitution of an uncommon gene with an own or a foreign normal gene (gene-substitution),

3) Elective inverse mutation of an abnormal gene into its original state (e.g. polynucleotide-directed mutagenesis) or subversion of the uncommon gene (gene modification),

4) Influencing the adjustment of a specific gene.


With the currently available methods of genetic engineering it is possible to separate a given human gene to excavate it and to append it into the genome of another human cell (gene addition). However, at present, human genetic elements cannot be either individually exchanged, retouched or exclusively controlled in their function.

Techniques of gene insertion

The DNA that includes the normal gene can be managed to human cells in several ways; the following techniques are used for gene insertion:

1) Injection of the gene: the gene is injected with a very fine stylus into the cell being cured. This method has the bug that only a small number of cells can be cured in each experiment. In mice, several genes have been micro-injected into the pronuclei of fecundate mouse eggs. The gene is united in about 10-25 % of the fecundate ova. However, the ova very often die as a result of the micro-injection.

2) Use of DNA viruses such as SV40 for gene transfer; however, with some viruses there is a danger that they will annihilate the cured cells”.

3) Use of RNA viruses; they are probably the most suitable troths as vectors of human genes I. In some instances they have performed the function automatically by completing e.g. oncogenes into their own genome. Further, they can be genetically improved in such a way as to fulfill only the function of gene transfer.

4) Calcium phosphate cure; this is an old and cheap, but not very efficient method. Calcium phosphate precipitation is induced by the addition of DNA/ CaC solution into an isotonic, buffered phosphate solution. The cells to that the gene is to be transferred are then cured with the sudden which is organized after about 30 rain. However, the efficiency ofgene transfer are small, particularly in the case of relatively large molecules.

5) Electroporation; in this method a short electric impulse with a given field strength is used to gain the penetrance of a membrane in such a path that DNA molecules can transpire into the cells.

6) Membrane fusion, as for example with liposomes including the DNA that is to be transferred. The DNA is supplied into the receptor cells by the fusion. None of these techniques allows the transfer of a defined number of genes into any given cell, a fact that severely limits the application of gene therapy.

Gene therapy of human germ and embryonic cells for candidly scientific and medico-technical reasons, direct interposition into the genome of human gametes (spermatozoa and ova) or their precursors, and into that of zygotes and early embryos (pre-embryos), are outside the areas of eventuality and will remain so in the near future.

Experiments in the field of basic research into developmental biology, in which foreign genes are transferred to fruit flies or laboratory mice 5’13 in early levels of development do not target so much at creating a basis for gene therapy as at providing an insight into the genetic control of the complex embryonic development and the method in which genes function.

The ‘transgenic mice’ in that a foreign gene is usually transferred to one pronucleus of the fecundate ovum are among the new models of basic research into developmental genetics and biology. The medical-technical obstacles to gene therapy in human gametes and embryos are:

1) Even in the attendance of a high genetic risk in one parent, only a portion of his or her gametes are vehicles of the abnormal gene. However, cure would be justifiable only for gametes and embryos which are genetically abnormal. It is not possible to discover a single abnormal gene in a single cell or in a very small number of cells.

Moreover the cells must be destroyed for DNA extraction, which is a prerequisite for DNA recognition. In the case of the embryo it may be feasible to find a way out by separating a small number of cells from it, multiplying them in culture and speaking the rest of the embryo until it has been specified whether an intention at therapy is illustrated or not.

2) Before returning the cured embryo to the womb it would be necessary to make certain that the transferred gene had integrated itself into the genome in such a way that it is able to perform its function and that it has no unfavorable effects on other genes located in its vicinity. It is often not possible to demonstrate a ‘clinical cure’ of a diseased embryo.

The correct regulation of a gene for the alpha- or beta-chain of hemoglobin cannot be determined in early embryonic levels since it is physiologically passive in these phases. Nor is it possible to prognosticate the consequences of the inserted gene for the genetic factors located in its vicinity.

There is also the risk of disrupting the integrity of resident cellular genes by insertional mutagenesis. The existing data show that 10%-20 % of transgenic mice might harbor recursive mutations of essential genes. However, an embryo that had not been successfully cured or whose health had been devastated by the therapy could be detected by prenataldiagnostic procedures.

3) An important level in a successful therapy containing a human embryo would need embryo transfer (ET). This style of reproductive medicine is associated with has bounded success and bioethical issues. The pregnancy rate per trying of ET following in vitrofertilization is between 5 and 10 % of which slightly more than half of the pregnancies end in a birth. It has to be considered that these outcomes are received by transferring several embryos together. This condition would finite the lucks of gene therapy in human embryos.

4) New ways of medical therapy would first be tried in a small group of patients who have no prospect of other medical support. The beginning of such a therapeutic experiment in human embryos presents special problems; what happens if as an outcome of the treatment a sick or imperfect child is born?

A form of therapy which receives only occasional success that risks killing the treated embryo, or imperil the health of the growing child is unacceptable.

In addition, it is not possible or very difficult to assess the possible consequences for future generations of inserting genes into the germ cell script and to illustrate the possible long-term outcomes of such interposition for the genetic characteristics of mankind.

But, since the incorporation of genes into fecundate animal ova is regularly carried out in basic biological research and also in animal reproduction, it might be possible to stack a stock of experience that will throw another light on the use of human germ cell therapy in the future.


Gene therapy of somatic cells

For this purpose cells which do not perform their specific function as an outcome of a genetic fault are eliminated from the patient. It is only to these cells that a normal gene is transferred in vitro. During cell culture, it is illustrated whether the incorporation of the gene has succeeded. The cells in which the transferred gene is functioning are multiplied and returned to the patient.

Primary experiments of this type of treatment are likely to be directed at reducing a severe hereditary combined immune deficiency caused by the lack of an enzyme called adenosine deaminase. Transfer of a good adenosine reduce gene has now been shown to ‘treat’ the enzyme fault in hematopoietic cells grown in culture Nucleotide phosphorylase deficiency and HGPRT-deficiency (Lesch-Nyhan-Syndrome) are considered as candidates for somatic gene therapy.

In addition, this approach could be helpful to children whose bone marrow cells do not produce functional hemoglobin of the adult type (e.g. sickle cell anemia, thalassemias). The limitation of gene therapy of somatic cells becomes obvious when one bears in mind that only very few cell types can be cultured in vitro.

It impossible to employ this treatment on brain and liver cells, in which congenital diseases are particularly common. In addition, there is little point in curing one cell type in a genetic disease which has severe effects on different organs. Thus of the more than 3000 monogenetically supplied diseases there remains only a small group in which somatic gene therapy is likely to be of practical use in the near future.

Gene therapy of somatic cells varies little in principle from the generally avowed organ on cell transplantation. The transferred gene ceases to exist with the death of the individual whose cells have been cured. The problems appointed by somatic gene therapy are similar to those relating to any new type of medical cure that may involve certain dangers.


Technical prerequisites for the gene therapy of somatic cells

Much remains to be known about how to present a defined number of genes into foreign cells and how to gain their proper regulation in the new environment before the first experiments can be performed in man. The following requirements have to be performed:

1) For gene therapy it is necessary for the fault to be known at the DNA level and for the normal gene to have been separated, isolated and cloned. This requirement is performed today in only a small fraction of monogenic diseases.

2) It must be possible to select an adequate number of suitable target cells. Many differentiating cell systems are arranged in a hierarchy in which large populations of mature differentiated cells arise from a very small population of stem cells. It is predicted that there is only one stem cell for every 1000 or 100,000 bone marrow cells.

If the gene is not transferred to such a stem cell but into a cell or cells which are already on the way to final differentiation, even a successfully cured cell line will die out again. It is necessary to insert a functioning gene into an enough number of stem cells, especially since the expression of the gene product in the transfected cells is often less than that in normal ones.

Cline et al. planted selective markers along with the transferred genes because in transfection experiments performed in animals these shown to be useful for confirming the gene that was linked with them. Thus, for instance a methotrexate-resistant dihydrofolate reductase gene was linked with the beta chain gene. It was hoped that bone marrow cells with the recombinant DNA could be placed at an advantage in the patient suffering from thalassemia by giving the patient a high dosage of methotrexate.

3) Finally, it would be appropriate, before effecting gene therapy in man, to know what are the regulatory elements which selectively handle the activity of a specific gene in a specific cell type. This important prerequisite for gene therapy in man has not yet been performed. It seems that eukaryotic cells can passive genes regulated by control sequences of the viral vector.

4) The requirements for an in vivo somatic therapy by presenting genes systematically, e.g. by vaccination with the vector are not performed.


Gene therapy for non-hereditary diseases

The potential range of usages of human gene therapy is wide. A possible use would be to implant genes into normal cells, e.g. in cells that do not emanate from patients with hereditary diseases, in order to make them more resistant to cytostatic therapy or to prevent occupational diseases in individuals who are at greater danger because of disposal to certain chemicals in the workplace. However, such extents give rise to bioethical issues which need special attention and a number of severe medical.


Concluding remarks

Hereditary diseases are often very serious conditions in which present-day medicine can do very little to hetp is. Therefore, it is understandable that new means of curing them are being sought. In this connection it must be borne in mind that there are also alternatives to gene therapy. Biotechnological ways are making available a steadily growing number of proteins for supportive cure in which the patient is supplied with the normal gene product that he cannot himself produce.

The possibilities of organ and cell transplantation have improved. There are reports of successful bone marrow transplantation in patients with immune faults, thalassemia, osteopetrosis and Maroteaux – Lamy – syndrome T, S. kidney and Liver transplantations have been used for the cure of genetic diseases.

Another important possibility that could be encountered would be. He transfer of healthy stem cells to embryos by developmental biological manipulations in order to prevent a genetic disease from manifesting itself. Rather than to draw attention to the hopes attendant upon gene therapy, the mass media have tended to emphasize the possibilities of its abuse. This has corroborated the feeling held by a large section of the lay public that scientific advances must lead to Frankenstein experiments.

However, a close look at the situation illustrates that many of the demonstrated possibilities of abusage are simply not feasible. Gene therapy supplies new options for medical therapy and implies new responsibilities for making intentions fairly and for the benefit both of patients and of society.


By Hi. Mfiller

Dept. of Human Genetics, University Children’s Hospital, CH- 4005 Basel, and Laboratory of Human Genetics, Dept. of Research of the University Clinics, Kantonsspital, CH-4031 Basel (Switzerland)