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X-linked severe combined immunodeficiency (SCID-Xl) is a rare human inherited disorder in which early T and natural killer (NK) lymphocyte development is blocked. The genetic disorder results from mutations in the common gammac chain that participates in several cytokine receptors including the interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15 receptors. We have shown in a previous report that gammac gene transfer into SCID-Xl bone marrow (BM) cells restores efficient NK cell differentiation. In this study, we have focused on the introduction of the gammac gene into SCID-Xl hematopoietic stem cells with the goal of obtaining differentiation into mature T cells. For this purpose, we used the in vitro hybrid fetal thymic organ culture (FTOC) system in which a combination of cytokines consisting of stem cell factor (SCF), Flt-3L, IL-7, IL-1, and IL-15 is added concomitantly. In this culture system, CD34(+) marrow cells from two SCID-Xl patients were able to mature into double positive CD4(+) CD8(+) cells and to a lesser degree into CD4(+) TCRbeta+ single positive cells after retroviral-mediated gammac gene transfer. In addition, examination of the output cell population at the TCR DJbeta1 locus exhibited multiple rearrangements. These results indicate that restoration of the gammac/JAK/STAT signaling pathway during the early developmental stages of thymocytes can correct the T-cell differentiation block in SCID-Xl hematopoietic progenitor cells and therefore establishes a basis for further clinical gammac gene transfer studies.

There are numerous inherited immunodeficiencies characterized by either defects in T, B lymphocytes, phagocytic cells or the complement system. About 20 genes involved in inherited immunodeficiencies have now been identified. This opens theorically the possibility to consider gene therapy for the most severe of the diseases. A logical approach consists in attempting gene transfer into hematopoietic stem cells in order to achieve a definitive cure. However, the presently available vectors, i.e. retroviruses induce only stable gene integration and possibly expression into cycling cells while most stem cells are in G0/G1. This precludes at this time efficient gene therapy for many inherited immunodeficiencies. Nevertheless in instances, where there is an early block in cell differentiation like in adenosine desaminase deficiency (ADA) or X-L severe combined immunodeficiency (IL2 R gamma deficiencies), a selective advantage could be provided to the few transduced stem cells enabling progressive lymphocyte differentiation. This hypothesis sets the basis for the ongoing clinical studies in patients with ADA deficiency and will be assessed in available animal model of XL SCID.