Update Summer 2000

Chairperson's Report: GENE THERAPY OF IMMUNE DEFICIENCIES

We are privileged to witness the work of Professor Alain Fischer and his team in Paris who have created a medical milestone for a significant group of children affected by primary immunodeficiency disorders.

Professor Fischer, we salute you and hope you wear our honour of you with pride.

Gene therapy consists in introducing a gene into the organism, in other words a DNA sequence with a therapeutic aim. This method can, in principle, be used for treating a large number of diseases, whether hereditary or degenerative (such as Parkinson’s disease or atheroma), or cancers. However, to this day, the technical obstacles are far from being all overcome, so that, despite important research efforts, tangible clinical results are still limited.

Of course, this therapeutic method can be applied to hereditary immune deficiencies once the gene of the specific disease is known. It is the case today for a large number of immune deficiencies, since we know the genetic origin of more than 40 hereditary immune deficiencies, especially those of the most severe forms.

Among these, the severe combine immunodeficiencies (SCIDs) constitute a "privileged" category for gene therapy, in the sense that these are very severe diseases, responsible for the death of children before the age of 1 to 2 in the absence of treatment. All these diseases are characterised by a blockage in the development of the T lymphocytes, major cells in the organisation of the immune responses against microorganisms. There are at least 7 different forms of SCIDs. The most frequent one is the X-linked SCID disease, characterised by an early blockage in the development of the T lymphocytes and of the Natural Killer lymphocytes (NK). This disease is caused by a mutation of the gene encoding the gc cytokine receptor. This molecule is expressed at the surface of all the cells originating in the bone marrow. Normally, the liaison of the cytokines to the receptor allows the development, at an early stage, of T and NK lymphocytes.

The classical treatment of this disease is allogeneic of bone marrow transplantation. When there is an identical HLA donor this therapy is efficient in most cases, so that there is no need for another form of treatment. On the other hand, in all the other cases (more than 8 patients out of 10) the result obtained by the bone marrow transplants is not satisfactory enough. This justifies the efforts to develop a gene therapy.

Six years ago, we decided to try to establish such a form of treatment, considering the severity of the disease (cf supra), and considering the fact that this disease constitutes a situation where the chances of success of the gene therapy are greater than those met in other diseases.

To transfer a gene to a cell, a vector, a transport system, is indeed necessary. The transport systems used at the moment are viruses. We take advantage of their natural properties to penetrate into the cells. The viruses available to treat cells of hematopoietic origin are called retroviruses. Retrovirus means that their genetic material is constituted of RNA and that it is transcribed back into DNA inside the infected cells.

These viruses, if they have interesting properties, have their limits. In particular, they are unable to allow the introduction of their genetic material within the nucleus of the cells which are not in. This is a major obstacle to the gene therapy of blood diseases. The total quantity of hematopoietic stem cells which we have at our disposal for the whole course of our life are not in cell division. They are in fact rebellious to this sort of treatment.

This rules out the use of gene therapy for a very large number of situations, in the present state of the available techniques. On the other hand, for the diseases of the lymphocyte precursors, this obstacle can probably be circumvented by the fact that the lymphocytes precursor cells are themselves in division and therefore potentially sensitive to these viruses. Once corrected, these cells can give birth to a very large number of lymphocytes, the life span of which is very long (several years).

So, a system of a globally average efficiency is susceptible to allow the correction, at least for a long period of time, of the T lymphocyte immune deficiencies, provided that the expression of the therapeutic gene produces a better growth of the cells thus corrected. This complex notion is however essential to the understanding of the applied strategy and obtained results.

We have accordingly constructed a retroviral vector containing the copy of the gene allowing the fabrication of the gc protein. This vector is produced by a cell called a packaging cell line (?) because it carries the viral proteins necessary to the making of the virus. The produced virus is not able to reproduce; therefore, it presents little danger for the human being. With the help of this viral preparation, we have shown that in vitro experiences it was possible to obtain the expression of the gc protein at the surface of the B lymphocytes of patients and to show that this protein was functional. The expression was stable during a long period of time. In the same way, it has been possible to correct in vitro the blockage of development of the T and NK lymphocytes by infecting cells in the bone marrow of deficient patients.

We have also created a model of mice deficient in gc protein by the technique known as homologous recombination. These mice have an immune deficiency close to the one of patients. We have treated these mice by ex vivo gene therapy by infecting the cells of the bone marrow by a vector containing the copy of the gc gene.

We have observed that that way it was possible to correct the immune deficiency of the mouse without causing any secondary effect. This observation has been carried out for a period of one year.
The majority of these very encouraging results have urged us to propose a clinical trial. This clinical trial has received the necessary authorisations. It was planned that 5 patients would be included in this trial which started in 1999.

The principle of this trial consists in taking bone marrow cells from the patients, purifying the precursors which express a molecule at their surface called CD34. Then these cells are cultured in the presence of cytokines to try to obtain a certain level of cell division, then they are infected 3 times 24 hours apart by the supernatant containing the retroviral vector. They are then washed and injected intravenously to the patients. This clinical trial is reserved to patients who do not have an identical donor in their family, and after of course obtaining a knowledgeable agreement from the family.

To this day, 5 patients have been treated. No secondary effect has been observed. The results are encouraging, since now in 3 treated patients, we have obtained a correction of the immune deficiency with a normalisation of the number of T lymphocytes, presence of NK lymphocytes and functioning of these cells. The results are still early for 1 patient, but they also seem encouraging. Therefore, 4 patients have been able to go home and 3 do not have any more treatment of any kind with the follow-up of more than one year for the first two patients. In one patient the treatment was less successful, likely as a consequence of an enlarged spleen.

Of course, we must wait to find out the duration of the correction obtained. It is not possible to exclude that later on the corrected cells disappear and that we have either to repeat the treatment. In any case, the result is conclusive and shows a possible efficiency of the gene therapy in favourable conditions. We are of course planning to pursue this trial by treating other patients.

What are the prospects for the future? Logically, it should be possible to consider a similar method for the other forms of SCIDs. We are now working on deficits in Rag1 and Rag2 which produce a blockage in the development of the T and B lymphocytes, with the hope that it will lead to a clinical trial in a year or two. Other forms of immune deficiencies where the gene deficiency is responsible for a blockage, at least partial, of the cell development, could probably be candidates for gene therapy, though the results then become more doubtful. It is the case for example for a T deficiency caused by a ZAP-20 protein deficiency, or maybe the Wiskott Aldrich syndrome. The development of new vectors having better results in infecting the hematopoietic stem cells will hopefully, however, make this technique more reliable in the next few years..