Retroviral Vector for Gene Therapy of X-Linked Severe Combined Immunodeficiency Syndrome

X-linked severe combined immunodeficiency syndrome (X-SCID) is a genetic disorder characterized by profound impairment of cell-mediated and humoral immunity. Affected children die of recurrent infections within 2 years of birth unless rescued by allogeneic transplantation from a suitable donor. Recently, the genetic defect responsible for X-linked SCID has been identified as a mutation in the γ chain of the IL-2 receptor, a protein also shared by the IL-4 and IL-7 receptors and therefore now denoted the common γ chain (γ_c). We report here the development of a high-titer amphotropic retroviral vector for transfer of γ_c. This vector was used to transfer a copy of the γ_c cDNA to murine 3T3 fibroblasts, CD34-enriched hematopoietic progenitor cells obtained from bone marrow and umbilical cord blood of normal donors, and to transplanted murine bone marrow progenitors. Murine 3T3 cells transduced by the retroviral vector were analyzed by Southern blot hybridization and Western transfer. Southern analysis confirmed the integration of unrearranged proviral DNA, and Western blot analysis demonstrated the expression of γ_c protein. CD34-enriched cells were infected with viral vectors bearing γ_c and grown in methylcellulose media. Individual colonies and pools of cells were analyzed 2 weeks later by polymerase chain reaction assay, which confirmed the proviral marking. The vector was also used to transfer a copy of the γ_c cDNA to murine bone marrow cells in a transplantation model. Infected marrow was transplanted into syngeneic Balb/c mice. Bone marrow and spleen samples from the recipients confirmed the presence of vector-marked donor cells in both tissues 3 months later. Thus, retroviral vectors can be engineered to transfer a normal γ_c cDNA to hematopoietic progenitors. These experiments have important implications for gene therapy for X-linked SCID.