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Gene Therapy
Santiago Grisolia, (Spain/Espagne)
President, Scientific Co-ordination Committee of the UNESCO Human Genome Project/
Président, Comité scientifique de coordination du Projet de l'UNESCO sur le génome humain
I.
INTRODUCTION
On September 14th of 1990 a little girl four years old, with a deficiency of adenosine
deaminase (ADA), was injected with 10º T cells containing the ADA gene. This was done in
the Children's Unit of Intensive Care of the Clinical Center of the National Institutes of
Health (NIH) in Bethesda, Maryland. That was the beginning of gene therapy in humans.
Although gene therapy is a rational and logical treatment for certain types of diseases in
humans, until rather recently the use of this type of technology was criticized as something
very far in the future and almost science fiction. As a consequence, there was very extensive
debate before the protocol for ADA deficiency was approved. Nevertheless, once this was
done, it impressed most people. Thus, in spite of the few people who suffer from ADA
deficiency in the whole world, the protocols used to correct this deficiency played a key role
in the demonstration of the feasibility of gene therapy, and established, therefore, the first step
towards a new medicine. The Advisory Committee of the NIH (RAC) has approved some 50
protocols for several diseases. Also France, Italy, Holland and China have started to use
some 10 protocols in quite a number of patients, as of the end of 1993.
II.
TYPES OF GENE TRANSFER
There are two main types of methods for gene transfer, those based on viral vectors
and those based on the introduction of DNA into cells or tissues by chemical or physical
means. There is work going on to develop other methods, such as the direct injection of
DNA, the formation of DNA-protein complexes with affinity for specific cell receptors, or the
transfer of DNA to tissues via accelerated particles. All these latter methods have, thus far, a
low transfer capacity, and/or result in rapid degradation of the DNA. On the other hand, the
viral systems are more effective, although they have the handicap of the transference and
expression of viral sequences. The main concern regarding the use of viral systems is
security. The design of the majority of the viral vectors is done in such a way as to minimize
the possibility of transfer of the complete virus and the possibility of gene recombination
which could give origin to the complete virus.
Viral Systems
They can be divided into those which adapt their sequences to the cell genome of the
recipient in a stable form and those which remain as episomes. The genomic integration has
the advantage that the therapeutic genes are inherited by subsequent cell generations.
However, they require cell division for their integration. The prototypes are the retroviruses.
Among the viruses which remain as episomes are the adenoviruses which have the advantage
of efficiently infecting non-dividing cells. However, since they do not become part of the
cell genome, and since they have been modified to inhibit replication, they are eliminated
when the cell divides. Because of the above considerations, a great number of gene therapy
protocols have used retroviral vectors. A great deal of effort has been spent in the
construction of these vectors so that they will be unable to complete themselves, otherwise
they might be able to cause a number of tumors. In these viruses, the packing of RNA in
virions is regulated by one sequence of the viral genome known as the packing signal. At
any rate, this is an area in which much is known but one in which much still needs to be done.
Non-viral Systems
Some of these systems are based on the synthesis of DNA-protein complexes which
may transfer via endocytosis the needed genes to a number of cells. However, these
complexes can suffer degradation by lysosomes. Also, these complexes cannot escape the
cell, therefore the efficiency is limited. Since many viruses which enter the cell have these
mechanisms, there are systems using co-internalization of adenovirus and DNA-protein
conjugates. These systems are much more effective and they are under extensive
investigation. The direct injection of DNA as a plasmid has been studied and it appears that
heart and skeletal muscle may be treated in this way. However, the main problem in humans
is the difficulty in maintaining a long-term expression.
III.
APPLICATIONS
Deficiency of adenosine deaminase
Deficiency of adenosine deaminase has already been discussed. However, at present
there are protocols using pluripotent stem cells from bone marrow which may really result in
cure of the disease, whereas thus far the treatment with retrovirus is only a palliative and
temporary treatment which necessitates periodic treatments.
Cancer
Among the variety of procedures directed to the treatment of cancer, it is important to
mention the technique known as "adaptive immunotherapy" that is the transfer of genes to the
immunological system to stimulate its function, or the transfer of genes to tumor cells to
facilitate their identification and to stimulate rejection. Other strategies are directed to
restoration of the normal phenotype in the tumoral cells via the transference of suppressor
genes or the improvement of bone marrow transplant for lymphoid tumors. The most recent
treatment is the induction of cell susceptibility via the use of the misnamed suicide genes.
1.
Adaptive immunotherapy. These procedures are directed to stimulate cell immunity
and are based on the insertion, in tumor cells or in lymphocytes, of cytokine genes. For
example, cytotoxic T lymphocytes (CTL) stimulated by IL-2 will fight foreign tissues. This
technique using LAC cells has been used to inject melanoma patients and has been partially
successful. In a few cases there was complete regression, particularly when the T
lymphocytes were obtained from the tumor. The use of the tumor necrosis factor (TNF) has
a significant effect on tumors. Unfortunately the dose needed is toxic for humans.
Nevertheless, the attempt has been made to obtain high concentrations in the tumor using
lymphocytes from the tumor treated with TNF. Another type of immunotherapy is based on
modification of tumor cells to produce cytokines or to express surface antigens. It has been
used for melanomas, and carcinomas from colon and mammary gland. They have use IL-2,
-4, -5 and -6 and the granulocyte stimulating factor CD-2 and interferon. All these
treatments are in progress and they appear to be more effective in primary tumor than in
metastases.
2.
Therapy with Tumor Suppressor Genes.
This treatment is theoretically possible
although difficult and far in the future. It has been tested experimentally for the restoration
of the suppressor gene of retinoblastoma (RB), in prostatic carcinoma, the p53 in mammary
carcinoma, in hepatomas and leukaemia. Although the technique inhibits cell growth, little is
known about the effect in vivo on a tumor. There are other difficulties because of the
complex character of tumor proliferation due to the implication of other genes (oncogenes)
and whether these techniques may be of use for all tumor cells.
3.
Cell Marking in Bone Marrow Transplant. Gene transfer can be very useful to follow
cells in vivo. Although bone marrow transplant is very useful, there are problems such as the
difficulty in obtaining histocompatible donors, and this has necessitated using autogenous
bone marrow, hopefully free of cancer, for some hematological conditions. Here is where
the marking could be useful to avoid the transplantation of bone marrow still containing
tumor cells, to separate stem cells or to locate the origin of a recurrence. There are several
protocols for myeloblastic leukaemia, other types of leukaemia, neuroblastoma, multiple
myeloma, and metastatic breast cancer.
4.
Induction of Cell Susceptibility.
This is known by the unfortunate name of the use
of "suicide genes". The procedure first tried consists in the introduction in tumor cells of the
thymidine kinase gene from herpes virus. The protein codified by this gene makes the cells
which contain it, susceptible to gancyclovir. The first application in humans has been the
treatment of brain tumors. Due to the absence of proliferative activity in the normal brain
tissue, the retroviral vectors will be integrated only in the genome of the tumor cells.
Interestingly, there is the so-called "by-stander effect", whereby even the tumoral cells in the
vicinity of those infected by the virus are also destroyed. Several variations of this technique
have been proposed using cytosine deaminase and 5-fluorocytosine. This combination
results in the production within the tumor cell of 5-fluorouracil, a well-known agent in cancer
chemotherapy. Another combination uses a gene derived from the varicela zoster virus and
anti-herpes drugs.
Cardiovascular Diseases
Genes can be transferred in vitro to the cells of the circulatory system, both endothelial
and those of the smooth muscle. The latter which are in a large number, have the peculiarity
that their proliferation can be regulated in vivo via the autocrine or paracrine production of
growth factors. The modified cells may be implanted in vivo either in a vascular transplant
or in a native artery. For the transplantation, the intimal layer of smooth muscle cells are first
abrased with an angioplastic globe. This results in a proliferation of the native cells to make
a new intima which includes cells modified by gene transfer. Thus far these treatments are of
very low efficiency. There are also additional problems regarding the effects of a gene on
cells of a different type.
Hematological Diseases
The manipulation of stem cells is essential to correct some hematological problems,
for example thalassaemia or when one requires a large amount of enzymes or proteins for
therapy. Since thus far gene transfer to stem cells of bone marrow is very difficult, the
co-expression of the gene of interest with a gene for resistance to multiple drugs (MDR) has
been tested. This gene codifies a protein which is able to pump out from the cell a number of
drugs which are toxic. There are some experiments using Taxol in murine models, which
need to be evaluated and extended before testing it in humans.
Lung Diseases
There are two of these which are of great actual interest: they are cystic fibrosis (CF)
and the deficiency of antitrypsin (AT). The main difficulty for the latter is that it is necessary
to obtain a blood level of approximately 2 mg/ml to avoid accumulation of elastase in the
neutrophils in the lung fluids which results in a progressive deterioration of the lung
epithelium. As for cystic fibrosis, the problem resides in the genetic codification of the
regulator of the transmembrane conductance. It is not yet known the type of the lung
epithelium in which the normal expression of the gene is needed or how to carry on a
selective transference. Because of the slow replacement of the respiratory epithelium,
several vectors derived from adenoviruses have been used instead or retroviruses. Thus the
genes for CF and AT have been transferred to the bronchial epithelium using adenovirus.
Although there are some problems and unknowns regarding concentrations and length of
treatments, the CF in humans is thus being treated. The first protocol used bronchoscopy but
the possibility of using aerosols is also being tested.
Liver Diseases
The liver possesses advantages for gene transfer because of its extensive circulatory
system. Although it has a great metabolic activity, it has a low proliferative capacity, thus
gene transfer via retrovirus is low. In tissue culture, hepatocytes may divide two or three
times which has permitted transfer with an efficiency of up to 30%. The first protocol for the
liver was started for the treatment of familial hypercholesterolemia (FH). FH is an autosomic
dominant problem caused by a defect in the gene for the receptor of the lipoproteins of low
density (LDL). The absence of the gene results in the accumulation of cholesterol, of LDL,
and then in atherosclerosis and coronary disease. The homozygote type cannot be treated by
diet or medication. After experiments in the Watanabe rabbit, primates were treated with a
retroviral vector which include the DNA complementary for the normal gene of the LDL
receptor plus a hybrid promotor to activate the transcription. After these studies in primates,
the protocols have been used in humans with good success.
Acquired Immune Deficiency Syndrome (AIDS)
Among the strategies used for AIDS there are protocols of gene transfer directed to
block the viral infection and/or to interfere with the expression of the genes. Although there
have been many variations of this, including the use of the so-called suicide genes in
CDH+lymphocytes, the results have thus far been disappointing.