Download Therapeutic Applications Stemming from Genetic

Therapeutic Applications
Stemming from Genetic Engineering
Rubén Lisker Y., (Mexico/Méxique)
President, National Genetics and Ethics Committee/
Président, Comité national de génétique et d'éthique
I.
1)
2)
3)
INTRODUCTION
There are three main types of genetic diseases:
monogenic, in which the presence or not of the disease is due to a single pair of genes
(called alleles). Some diseases require that both alleles be abnormal in order for them
to appear (they are called recessives) while only one gene needs to be abnormal for
others to be present (dominant). They all have characteristic family trees, and
although the individual frequencies of most of them is quite low, more than 4,000
diseases have been described and together they constitute a significant health problem;
polygenic, in which the disease is due to the additive effect of several genes and
environmental factors play a role in their presence. There are no typical family trees
and what is seen is familial aggregation of the condition. Some common diseases as
the congenital malformations, diabetes and hypertension are a good example of them;
and,
chromosomal, which can be defined as those where a microscopically visible
chromosomal abnormality is present.
Gene therapy should be most useful in the monogenic diseases, and it may take longer
to design a way to approach the polygenic disorders. In order to better understand the
significance of future gene therapy for genetic conditions it is best to review what is being
done at present, which falls into the general area of environmental manipulation.
II.
ENVIRONMENTAL MANIPULATION
Substrate Restriction
It is useful in situations in which the disease is due to the accumulation of a substance
which is not adequately metabolized by the organism.
A classical example is
phenylketonuria. It is inherited in an autosomal recessive fashion and the affected children
are born normal but quite rapidly have slow psychomotor development and other difficulties
which lead to profound mental retardation as the main problem. The disease is due to the
accumulation of an essential amino-acid called phenylalanine and several of its metabolites,
due to the absence of the enzyme phenylalanine hydroxylase. Phenylalanine is not
manufactured by the organism but it is ingested in different nutrients, basically milk, and it is
enough to substitute in the diet, regular milk with phenylalanine free milk to avoid the clinical
manifestations. For the treatment to be successful it is important to initiate it before the child
is one month old, and the diet should not completely lack phenylalanine, as some is needed to
allow a normal development.
Administration of a Missing Substance
When a genetic disease is due to the absence of a substance normally produced by the
organism, its administration can be a very effective treatment. A very good example is
Gaucher's disease type 1, inherited as an autosomal recessive and the main abnormalities are a
vary large spleen, bone lesions and blood cytopenias. It is due to the deficiency of the
enzyme acid beta-glucosidase, which is now possible to extract from placental tissue, and give
it periodically to affected individuals with excellent therapeutic response.
Unfortunately, there are many other disease which can not be treated in this manner
because there is no natural source of the deficient substance nor can it be synthesized, besides
being unable to introduce it to the relevant tissue. To solve these types of problems, organ
transplantation has been tried successfully in a few diseases: familial hypercholesterolemia,
Fabry’s disease.
Administration of Supplementary Coenzymes
The coenzymes are substances that are capable of activating certain enzymes to
perform their metabolic functions. Some genetic diseases can be corrected by the
administration of the relevant coenzymes, almost always in very high doses. One example is
the use of vitamin B6 (pyridoxine) for the treatment of certain types of anaemia.
Avoidance of Dangerous Substances
There are several genetic diseases in which the affected patients have abnormal
symptoms only when exposed to certain foods or chemicals. A good example is red cell
glucose-6-phosphate dehydrogenase deficiency. This enzyme deficiency is the most
common metabolic genetic disease present in humans. It affects mostly males and the
deficient individuals are generally normal except when they ingest certain chemicals such as
the anti-malarial primaquine, or food such as broad beans, which produce massive red cell
destruction manifested by acute anaemia, jaundice and other symptoms, which can be avoided
by not using agents which are known to produce the haemolytic crisis.
The Case of Folic Acid
A recent finding of great interest is that the administration of periconceptional folic
acid can prevent the appearance of neural tube defects, in particular anencephaly. The oral
use of this vitamin for 2 months before and after conception reduces the frequency of
anencephaly in 70% in high risk groups (couples who have already had one child with this
malformation).
III.
DNA MANIPULATION
We are at the beginning of DNA manipulation for the treatment of genetic diseases,
but the future is extremely promissory. In theory, it can be done in at least two manners:
inducing gene activity and by genetic engineering.
Induction of DNA Activity
In some micro-organisms the existence of regulatory genes of genetic activity has been
proved, and it is possible to induce the production of certain enzymes by the addition of the
appropriate substrates. It is not known if in man the same is true, although there are some
indications of this possibility. Gilbert disease is characterized by mild jaundice since
childhood and it is due to the deficiency of a liver enzyme called glucoronyl transferase. Its
activity can be induced by the administration of sodium barbital, which makes the jaundice
disappear.
Genetic Engineering
Genetic engineering is the group of procedures that allow the modification of the
genetic material, DNA. There is great hope that these technologies will be of substantial
benefit to humankind.
a.
DNA recombination.
To recombine DNA, it is first fragmented by the use of
the so called restriction enzymes, which recognize specific short sequences in DNA. The
fragments can be put together as desired, including the joining of DNA from different species.
The procedures allow the introduction of a gene that codifies for human anti-haemophilic
globulin, for example, into the DNA of a live organism like a bacteria, in order for it to
synthesize this substance. It is possible to make true bacterial factories that produce a variety
of human proteins, for instance insulin, which is used for the treatment of diabetes mellitus
and various others.
An interesting point of recombinant DNA research is that there is concern that the
"new" life forms produced by this technology could have negative characteristics, the so
called Frankenstein factor, that could release into the environment very small and dangerous
"monsters", capable of great harm. In the 1970’s the scientific community produced a set of
rules for the laboratories working in this type of research, in addition to stimulating a long and
difficult discussion on the desirability of recombinant DNA research, which could alter the
natural order (divine or otherwise) by creating artificial forms of life. There was no
unanimous accord but most scientists believe that the possible benefits greatly outweigh its
potential risks. The experience so far seems to strongly support this belief.
b.
Substitution of human genes.
It seems logical that to try this form of therapy in
humans, the answer to three questions are needed:
•
is it possible to introduce the desired genes to the target cells?;
•
can the gene activity be properly regulated so that the disease is cured?; and,
•
can we be sure that the procedure will not damage in the short and long run the
individuals treated?.
The answer to the last question may take a long time regarding such fields as cancer
development, fertility rate, etc..
There are two types of cells that can be treated: somatic and germline. In the first
case whatever benefit or difficulties are obtained, they affect only the patient being treated
and pose no particular ethical questions, and can be consider in the same manner as any
experimental human treatment. On the other hand, germline manipulation can affect not only
the individual treated, but all his or hers progeny, rapidly multiplying the potential harm or
benefit. Many people believe that this should only be done, if at all, after having a vast
experience with the results of somatic gene therapy.
Bone marrow transplantation.
Genetic diseases can be treated by transplantation of
normal allogeneic (same species) bone marrow or autologous (self) bone marrow into which
by genetic engineering (gene therapy) a normal gene has been inserted. This approach seems
logical in the treatment of diseases that affect the haemopoietic tissue as it can be expected
that the transplanted normal genes will be properly expressed and regulated, and in fact good
results have been obtained in the treatment of beta-thalassemia with the transplantation of
allogeneic HLA-identical normal donors.
Gene therapy.
The first successful experience in humans was with a 5 years old girl
suffering an immune deficiency due to the absence of the enzyme adenosine deaminase
(ADA). This abnormality lead to the destruction of the T lymphocytes of the patients
immune system, leaving her very vulnerable to numerous infectious agents. The treatment
consisted in:
i)
the extraction of T lymphocytes from the patient;
ii)
the introduction to the genome of the extracted lymphocytes of a normal ADA
gene using, as a vector, a retrovirus;
iii)
the engineered lymphocytes were cultivated to increase their number; and,
iv)
the cells were transfused back to the patient.
Two years after treatment the patient was fine with acceptable ADA levels although
treatment had to be repeated every 6 months. At present, most gene therapy protocols deal
with cancer treatment utilizing several different strategies and soon information will be
available on their efficiency.
c.
cloning.
Somatic cells have a complete chromosome complement, identical to
the one of the zygote, but as a result of cellular differentiation different genes are functional in
the various tissues and organs. If it were possible to dedifferentiate somatic cells so that they
could act as zygotes, it would be possible to start their development as independent embryos
and finish their growth in surrogate mothers and eventually in test tubes. Every subject
would be identical to the original donor and there would be no limit to the number of them
that could be produced.
Cloning has been possible in frogs by introducing the nuclei of their intestinal cells
(somatic cells) to ova of the same species that have had their nuclei surgically removed
previously.
The hypothetical and extremely remote possibility of obtaining by cloning a human
being has inspired at least 2 novels, one called “The Sons of Brazil”, which is science fiction,
and the other purported to describe a real event and is entitled “In his Image: The Cloning of
a Man”. The latter was proved to be a hoax, but it is interesting to discuss whether this
would be beneficial. We believe that the results would be impossible to predict because of
the environments influence (which is not reproducible) in the physical, intellectual or
character development. Einstein and Freud were very distinguished intellectuals whose ideas
enriched humankind, but both were a product of their times. Who can predict what would
happen if today we could fabricate by cloning 1.000.000 Einsteins, who would all be eminent
physicists? Would humanity benefit?