NFP37 SOMATIC GENE THERAPY Divulgation Abstracts phase B

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HERRERA, Pedro Luis	Dep.de Morphologie, CMU, Faculte de Medecine, 1 rue Michel-Servet, CH-1211 Geneva 4 tel 022 702 5225; fax 022 702 5260 e-mail: Pedro.Herrera@medecine.unige.ch
Title:	Devising an approach to modulate the graft versus host disease in protocols of adoptive immunotherapy
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DIVULGATION TEXT AT SUBMISSION (1998):

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Graft versus Host Diseases (GvHD) and conditional expression of genes

One of the main drawbacks of bone marrow transplantation (BMT) to depleted patients is the risk of developing a GvHD, either acute or chronic that in some cases can even result in a fatal outcome. The GvHD is due to the presence of donor T cells within the bone marrow graft. Nevertheless, the perfusion of T cell-depleted marrows to leukemic individuals is frequently ineffective because there is an increased risk of graft rejection, of tumor relapse, and of developing viral infections or secondary malignancies. In addition to that, patients presenting relapse of primary disease after allo-BMT, do recover when treated with activated donor T cells; such treatment is termed "adoptive immunotherapy". The presence of activated donor T cells has therefore a number of advantages, provided these cells can be deleted in case of a severe GvHD. It is thus mandatory to develop the therapeutic tools that should allow the modulation of GvHD.

Recent studies, including clinical trials, have demonstrated the feasibility of the project that constitutes the object of the present application. Indeed, it is now possible to isolate T cells that can be genetically modified ex vivo without diminishing their functional capabilities. In particular, one study reports that GvHD can be effectively controlled by gancyclovir in patients having received donor T cells that had previously been transfected with a suicide transgene encoding the herpes simplex virus thymidine kinase (HSV-TK). Similar results have been observed as well using transgenic mice bearing a HSV-TK transgene.

A new development of the Cre-loxP system allows, in vivo, the exquisite temporal control of the recombinant activity of Cre in selected cells. This consists in a fusion enzyme, Cre-ERT, in which the binding domain of a mutated estrogen receptor (ERT), that solely recognizes the anti-estrogen 4-hydroxytamoxifen (4-OHT), has been added to Cre. In our conception, the Cre-ERT gene, flanked by two loxP sites, would interrupt another gene of interest whose expression should occur only at a particular time point (Figure 1). The originality underlying our proposal is that the Cre-ERT coding region would be the block of the transcription of a gene placed downstream and simultaneously the inducible and irreversible activator of the transcription of this very same gene. Such a control of gene activity would be of obvious interest for genes having therapeutic properties.

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Figure 1. The Cre-ERT gene, surrounded by two loxP sequences, is placed between a tissue-specific promoter and the coding region of a gene whose expression needs to be temporally controlled. The Cre-ERT produced by a cell having such a transgene is inactive for it is forming a cytoplasmic complex with an inhibitory protein. Administering 4-OHT frees the Cre-ERT recombinase, which becomes then active and nuclear. Its activity catalyzes the excision of its own gene, the Cre-ERT gene, which is floxed, and results in the transcription of the downstream sequence.

The proposed approach should permit the selective ablation of the GvHD-inducing activated T cells, simply by providing a pharmacological substance (4-OHT) that, in addition, has no major side effects. This will be achieved by introducing ex vivo, in the CD4+ and CD8+ of the donor, a transgene encoding the A subunit of the diphtheria toxin (DT-A). DT-A adenoribosylates EF-2 (elongation factor-2), thus inhibiting protein synthesis, which in turn results in cellular death. The toxic effect of DT-A is cell autonomous, for neighboring cells remain unaffected. The transgene will be designed so that a T cell-specific promoter (Lck) is controlling DT-A expression, but with a loxP-flanked Cre-ERT gene located in between. DT-A synthesis should occur in the transfected Iymphocytes only upon administration of 4-OHT to the patient.

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