Therefore, the human c might function less effectively as a mouse IL-15 receptor subunit than as a mouse IL-7 receptor subunit. less likely to be located within or near transcriptional start sites than RV vector integration sites. To evaluate the therapeutic efficacy, bone marrow cells from c-knockout (c-KO) mice were infected with the FV vector and transplanted into c-KO mice. Transplantation of the FV-treated cells resulted in the successful reconstitution of functionally active T and B cells. These data suggest that FV vectors can be effective and may be safer than conventional RV vectors for gene therapy for SCID-X1. Introduction X-linked severe combined immunodeficiency (SCID-X1) is usually a GBR-12935 2HCl life-threatening immunodeficiency disorder, characterized by defective T and natural killer (NK) cell production and the development of functionally impaired B cells that Rabbit Polyclonal to GPR150 lack the capacity to produce immunoglobulins. These defects result in a profound reduction in the development of both cellular and humoral immunity. SCID-X1 is caused by inactivating mutations in the gene encoding the cytokine receptor chain (c), a common subunit of the receptors for interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21 [1]. Bone marrow transplantation (BMT) from human leukocyte antigen (HLA)-identical siblings can cure the disease with a success rate of approximately 90%. However, BMT from non-HLA-identical donors results in lower survival rates due to a high risk for complications such as graft-versus-host disease, graft rejection, and incomplete T cell engraftment [2], [3]. Consequently, gene therapy approaches have been developed as an alternative treatment option for those patients lacking appropriate donors. The first gene therapy clinical trial for SCID-X1 was carried out by a French group in 1999 [4], [5]. In that study, a conventional retroviral (RV) vector expressing c was used, and resulted in the reconstitution of T and NK cell populations, and the recovery of humoral immunity. However, over time, acute T cell leukemia developed in 5 of the 20 patients receiving the therapy. The leukemia cells of these patients showed aberrant and high expressions of proto-oncogenes such as analysis of foamy computer virus vectors.(A) Structure of the foamy computer virus vectors. The UCOE631 promoter sequence from the human HNRPA2B1-CBX3 locus and transgenes were inserted into the FV vector. (B) Cell-surface expression of c on ED40515(C) cells transduced with the FV-IL2RG vector. (C) STAT5 phosphorylation upon IL-2 stimulation. ED40515(C) cells, an ED40515(C)-derived transfectant with a c gene, ED cells, and ED40515(C) cells transduced with the indicated vectors were stimulated with IL-2 for 30 min, and STAT5 phosphorylation in each cell GBR-12935 2HCl line was detected by a STAT5 phosphospecific mAb. FV particles were produced by transfecting 293T cells with the resultant gene transfer vector plasmids and three helper plasmids (pCiGS, pCiPS, and pCiES) using GBR-12935 2HCl FuGENE HD (Roche Applied Science), as previously described [13]. Culture supernatants were harvested after 48 hours and concentrated by ultracentrifugation. A c RV vector was constructed by inserting the human c cDNA into the multi-cloning site of pMX-IRES-EGFP. Retroviral particles were produced by transfecting into amphotropic retrovirus packaging cells (PLAT-A cells) with pMx-IL2RG-IRES-EGFP using FuGENE HD. Culture supernatants made up of the RV vector particles were harvested after 48 hours. Cell lines A human T cell line, ED40515(C) [20], which lacks c expression, and an ED40515(C)-derived transfectant with a c gene, ED, were described previously [21], [22]. These cell lines were cultured in RPMI1640 medium supplemented with 10% FCS. Mice The c-KO mice were previously reported [23]. c-KO mice on a NOD/scid background [24] were obtained from the Central Institute for Experimental Animals (CIEA, Kawasaki, Japan). They were housed under specific pathogen-free conditions in individually ventilated cages and supplied with sterile food, water, and bed linens. All procedures were performed according to protocols approved by the Institutional Committee GBR-12935 2HCl for the Use and Care of Laboratory Animals of Tohoku University (2011MA139). Vector transduction and BMT Lineage marker depleted (Lin?) cells were purified from the bone marrow cells of male c-KO mice using.