Biological Ethics

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Chapter 20. Technology Genetics applied to humans

N. López Moratalla and E. Santiago

a) Human recombinant DNA therapy

Gene transfer

issue A very large number of diseases - some 2,500 - have their origin in a genetic defect - alteration of a gene or its expression system - which does not allow the synthesis of a certain protein in its biologically active form. In the future, patients with these anomalies may be able to benefit from the techniques described in the previous chapter. Therapy Genetics refers to the possibility of introducing DNA to replace that of the affected genes in order to correct their alterations.

Enzyme replacement therapy, encoded by the affected gene, could correct the various enzymopathies; however, since enzymes are primarily intracellular, this is very difficult, as they do not penetrate cells. Attempts have been made to inject the enzyme directly, either free or contained in microcapsules, erythrocytes or liposomes, as well as bound to physiological transporter molecules.1 2 Efficacy is limited by the relatively short lifetime of the enzyme, both in circulation and in the intracellular environment, and also by the immune reaction to foreign proteins in the body. Moreover, this therapy does not prevent the alteration Chemistry from being passed on to subsequent generations.

An enzyme defect can lead to the accumulation of a metabolite such that tolerable values are exceeded - with serious consequences if in the brain - or to a lack of a product that can be severely limiting; it is not always feasible to remove or provide such a metabolite. There are already many techniques that allow prenatal detection of genetic diseases, in cell cultures of amniotic fluid obtained between 14 and 17 weeks of gestation, programs of study chromosomal and metabolic samples of chorionic tissue from the 9th week and also the study of cell-free amniotic fluid where metabolites appear as a result of alterations in metabolic pathways. The massive detection of those diseases that are susceptible to treatment - through the use of certain diets - has allowed thousands of affected children to reach a completely normal neurological development . These diseases include phenylpyruvic idiocy, congenital hypothyroidism, or congenital biotinidase deficiency. In certain cases, an appropriate per diem expenses can address these situations.

The solution final for diseases that have their origin in an alteration of a single gene may lie in a therapy Genetics that allows the insertion of the defective gene, or the correction of the signals that control its expression in the affected tissues. These "corrections" could theoretically be carried out in the gametes of the progenitors, in the zygote or early embryo; also in the adult, either by infection with a viral vector from which the pathogenic character has been removed, or by transplantation of cells previously corrected in culture. On the other hand, the recently designed "gene making machine" allows the automatic synthesis of DNA fragments with prefixed sequence; this makes it possible to "comfortably" dispose of genes or control consequences to modify expression and above all in the necessary quantities for transfers.

In July 1980, Cline transplanted bone marrow cells, corrected in culture by introduction of the -globin gene, into two patients with severe thalassaemia, without a positive result . The realisation of this therapeutic essay has been highly criticised, as there was a lack of experimental data in animals to support the attempt with a minimum of guarantees. However, this technique offers future possibilities for correcting defects in easily proliferating tissue cells. Suitable vectors are already available to exert a selective pressure on the proliferation of the corrected cells. On the other hand, the conditioning factors, such as location on the chromosome, position and proximity of certain signals, which can affect gene expression, are unknown, and even more so are the means to direct the introduction of the DNA fragment at precise locations on a given chromosome.

In 1979, Fleischman and Mintz3 successfully carried out a correction Genetics, in homozygous mice carrying a mutation at the W site, which results in anaemia, sterility and lack of pigmentation. The cells of the blood line develop from day 11 of the embryonic development and the anaemia-causing effect appears on day 13, when the liver tissue becomes the main producer of red blood cells. These authors injected normal liver cells, via the transplacental route, between days 12 and 13 into embryos with this deficiency and non-anaemic mice were born. More recently, the expression of the human insulin gene in specific tissues in response to its inducers - glucose, amino acids, even the hypoglycaemic agent tolbutamide - has been achieved in mice born after microinjection of the gene in the embryonic phase4.

The greatest prospects are presented by diseases caused by genes whose control is not specific and therefore are expressed in many cell types. These include Lesch-Nyhan disease, in which mental retardation and self-mutilation are observed, caused by deficiency of the enzyme xanthine-guanine phosphoribosyl transferase, and two severe immunodeficiencies caused by damage to the enzymes purine nucleoside phosphorylase and adenosine deaminase. The gene manager for Lesch-Nyhan disease was transferred to the mouse as early as 19875.

It is also thought possible to transfer the genes for dihydrofolate reductase, which confers resistance to methotrexate, an anticancer drug that is highly toxic to actively proliferating tissues such as bone marrow, to the bone marrow of cancer patients. Thus, by increasing the concentration of the enzyme inside "labile" cells, it would have been possible to make them "resistant" to methotrexate.

Induced mutagenesis

The effects of gene introduction on the homologous sequence of a chromosome have recently been studied in mammalian cells. Injection of DNA into the nucleus has been shown to produce, quite unexpectedly, a high frequency of mutation in the cognate gene. This offers the potential power to manipulate genomes of higher organisms6.

Thomas and Capecchi7 have studied in rat cells the mutated gene - "neo" gene - whose normal expression confers resistance to the toxic G418. This is an "amber" mutation that causes premature termination of the protein. They have corrected this gene by microinjection of a different form of this same gene, also mutated by a deletion that removes the last 52 amino acids of the protein. The defective genes have result corrected by compensated mutation. These genes retain the original mutation, but acquire another one that compensates for the defect. The frequency of this phenomenon is very high and depends not only on homology but also on the anomalous pairing between pairs of instructions of the introduced DNA and the corresponding sequence of the one that was part of the genome. The existence of repeated sequences facilitates this process.

There are several aspects to consider in the application of this technique. Firstly, great care needs to be taken when manipulating mammalian genomes by gene transfer, as it is possible to induce undesirable mutations when attempting to correct a gene. These observations suggest, on the other hand, new methods to induce, for example, through a plasmid, mutations in specific regions such as promoters, cis-acting regulatory sequences. In other words, mutations in specific areas, which could perhaps be used to modify gene expression.

evaluationethics

In the case of human experimentation, it is necessary for the researcher to have sufficient scientific skill , to want to intervene for therapeutic reasons and to have carried out the previous experimental work on animals with sufficient breadth and depth, a point that has been adequately underlined in the Helsinki Declaration of 1975. Due to the B complexity of the problems and variables involved in the planning of human experimentation, not only the codes of ethics, but also the civil laws of some States now require that proposals for experimental programmes be drafted and submitted to the criteria of the commissions set up for their study, review and approval. The same codes also require that the experimental programme be preceded by a careful study of the foreseeable risks, which must be prudently assessed in relation to the expected benefit to the subject, or to others. This last condition "or for others", contained in the Helsinki Declaration, has been attenuated in its dangerousness by a paragraph introduced in 1975, not foreseen in the 1964 essay , and which clearly states that the interests of the subject must be placed above the interests of science and society.

At the present time, the application of Genetics therapy to humans requires prior programs of study in animals, which at least ensures the following aspects8 9:

First, the gene to be introduced must penetrate the cells and remain. Penetration seems to be feasible, at least in cells that are relatively easily available, such as bone marrow, liver or fibroblasts, using retroviral vectors carrying the normal gene. We have already mentioned that insertion into the chromosome and its stability in the chromosome is a problem that still requires further work programs of study.

Secondly, that the gene is expressed and that this expression can be regulated in such a way that the speed of training of the product is appropriate. We have already seen that not all introduced genes are expressed and the current knowledge on eukaryotic gene regulation is still insufficient.

Finally, it is necessary to ensure that the presence of the new gene, and that of the DNA associated with it and facilitating penetration, does not damage the recipient cells or produce mutational effects in the genome, problems that still require long-term observations deadline. The effects of a gene in the wrong place on a chromosome, and how it may influence the genes next to it, are unknown. There is also uncertainty about whether retroviruses used as vectors can become pathogenic in recombination.

The correct transfer of a gene into a eukaryotic cell is no easy matter; in fact, it has never been possible to identify genes separately by their presence, but rather to recognise them by their absence. These limits and uncertainties are continually reflected in the scientific literature10 11 12 13.

For the time being, therefore, therapy experiments Genetics seem premature, whatever the method employee, transplantation of corrected cells, microinjection into embryo cell nuclei, etc. The experimental nature of an intervention is not a reason for absolute prohibition, from an ethical point of view, and can be acceptable, even with serious risk, if it has a therapeutic character; however, in the current status , the therapy Genetics would have a net character of research - with very little chance of therapeutic success - and therefore, for the time being, should only be carried out on animals. Even more experimental would be Genetics therapy applied to humans in the embryonic phase, where the chances of success are even lower due to the great fragility of the embryo and the obvious technical difficulties of making a sufficiently early diagnosis.

At final, there will be no ethical subject obstacle to apply the therapy Genetics once the previous research stage has been passed in the animal. In the event that correction at the embryonic stage is necessary, the technique must be sufficiently advanced so as not to expose the embryo to probable death. It is obvious that the lawfulness of these interventions on the human genome - or on any of its cells - presupposes that the purpose is strictly therapeutic, that is to say, understood as the correction of a disease, of an anomaly Genetics. This intervention subject should never be the result of any other desire which, given the WHO definition of "health" as "total physical, intellectual or social well-being", could erroneously be considered as a therapeutic aim. The use of this technology for eugenic purposes will be discussed below (cf. chapter 22).

b) Some applications of DNA sequencing

The individual specificity of certain DNA sequences means that once they have been detected, they can be used as individual genetic markers, as genuine "fingerprints" in the genome. These genetic markers are determined even before organ transplantation and at programs of study on the spread of tumours.

The use of these markers for parentage identification offers a much higher accuracy than hitherto standard techniques. For example, the high polymorphism of satellite DNA in the globin gene region makes it possible to establish maternity with certainty. Likewise, these markers make it possible to establish paternity with high certainty. For this reason, they are already being used in some countries to settle immigration problems of individuals claiming kinship. They have also begun to be used in forensic laboratories14 15 16.

The use of this technology for the purpose of identifying kinship is obviously lawful in itself; however, it is to some extent an expression of a strong will to dominate the individual, in that it implies the desire, which can be taken to the extreme, for a programming of human society, where individual freedom, and even the right to privacy, can be reduced to a secondary level in the face of the will to plan, on the part of those in power.

Notes

(1) BRADY, R.O., TALLMAN, J.F., JOHNSON, W.G., GAL, A.E., LEAHY, W.R., QUIRK, J.M. and DEKABAN, A.S. "Replacement therapy for inherited enzyme deficiency. Use of Purified Ceramidotrihexosidase in Fabry's Disease". N. Engl. J. Med., 289 (1), 9, 1973.

(2) BRADY, R.O., PENTECHEV, P.G., GAL A.E., HIBBERT, S.R. and DEKABAN, A.S. "Replacement therapy for inherited enzyme deficiency. Use of Purified Glucocerebrosidase in Gaucher's Disease". N. Engl. J. Med., 291, 989-993, 1974.

(3) FLEISCHMAN, R.A. and MINTZ, B. "Prevention of genetic anemias in mice by microinjection of normal hematopoietic stem cells into the fetal placenta". Proc. Natl. Acad. Sci. USA, 76, 5736-5740, 1978.

(4) SELDEN R.F., SKOSKIEWICZ, N.J., HOWIE, K.B., RUSSELL, P.S. and GOODMAN, H.M. "Regulation of human insulin gene expression in transgenic mice". Nature, 321, 525, 1986.

(5) HOGAN, B. "Engineering mutant mice". Nature, 326, 240, 1987.

(6) LISKAY, M. "Manipulation just off target". Nature, 324, 13, 1986.

(7) THOMAS, K.R., and CAPECCHI M.R. "Introduction of homologans DNA sequences intro mammalian cells induces mutations in the cognate gene". Nature, 324, 34-38, 1986.

(8) ANDERSON, F. and FLETCHER, J.C. "Gene therapy in human beings: when is it ethical to begin? N. Engl. J. Med., 303, 1923-1926, 1980.

(9) MERCOLA, K.E. and CLINE, M.J. "The potentials of inserting new genetic information". N. Engl. J. Med., 303, 1298-1230, 1980.

(10) KOLATA, G.B. and WADE, N. "Human gene treatment stirs new discussion". Science, 210, 407, 1980.

(11) ANDERSON, W. "Prospects for human gene therapy". Science, 226, 401, 1984.

(12) MOTVLSKY, A.G. "Impact of Genetic Manipulation, Society and Medicine". Science, 219, 135, 1983.

(13) DAVIS, B.D. "The two Faces of Genetic Engineering in Man". Science, 219, 1381, 1983.

(14) DNA fingerprinting. "DNA probes control immigration'. Nature, 319, 171, 1986.

(15) NEWMARK, P. "DNA fingerprinting at a price at ICI'S UK laboratory". Nature, 327, 548, 1987.

(16) JEFFREYS, A.J. "Highly variable minisatellites and DNA fingerprints". Biochem. Soc. Transactions, 15, 309-317, 1987.