Following extensive preclinical work, a clinical trial was performed between 1999 and 2002 in 10 patients suffering from SCID (Severe Combined Immune Defi ciency)-X1 disease caused by c gene mutation. It was based on the ex vivo transfer of the c gene into CD34(+) bone marrow cells by using a first-generation, LTR-competent, non-replicative Moloney retroviral vector. This led to sustained correction of the T cell immunodeficiency and clinical benefit in 9 patients with a follow-up of 6 to 10 years. The trial was suspended following occurrence of a leukaemic disease in 4 subjects (reversible in 3) as a consequence of LTR-driven, aberrant expression of an oncogene close to the location at which the retrovirus had integrated into the genome.
A new SCID-X1 clinical trial (based on the use of a self-inactivated retroviral vector) will be implemented. Predictive toxicity assays for oncogenesis are being generated by using p19 arf-deficient, cancerprone mice crossed with the relevant immunodefi cient models (c-,Rag-1- Artemis-deficient) for gene therapy with the vectors to be tested before their clinical application. Long-term assessment of both clinical and experimental models of SCID gene therapy provides setting for analyzing the dynamics of T cell populations, based on a combined high-throughput analysis of the retroviral integration sites (clonal signature) with the TCR repertoire and T cell subset phenotype.

We have characterized a population of human lymphocyte progenitors that can differentiate into T, B and NK cells and that is detectable in the cord blood, bone marrow, adult blood and thymus. Preliminary data indicate that lymphocyte progenitors can be both extensively amplified (x 80) and specified toward the T cell lineage in vitro on stromal cell lines expressing the delta 1 Notch ligand. Our aim is to validate this observation, further characterise those cells optimize expansion conditions both in vitro and in vivo in xenogeneic murine models and study the migration capacity and ability to differentiate into mature functional T cells. This would constitute a prerequisite for potential clinical application, by speeding up T cell reconstitution in allogeneic HSCT or gene therapy.

We have recently identified the gene mutation responsible for a form of SCID. This SCID form is particularly interesting because it is the only form of SCID with profound lymphopenia (i.e. T- NK-) associated with (i) an early maturation arrest in the myeloid lineage at the myeloblast-promyelocyte stage and (ii) bilateral sensorineural deafness. Our results suggest a novel mechanism regulating energy metabolism and/or controlling apoptosis and which is involved in one of the most severe human immunodeficiency syndromes. Our goal focuses on understanding the mechanism of action of AK2 in haemato/lymphopoiesis based on the silencing of AK2 expression by SiRNA and the generation of conditional KO mice.

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