344. Application of a suicide Gene to X-SCID Gene Therapy

Abstract

X-linked severe combined immunodeficiency (X-SCID) is a fatal disease characterized by the absence of humoral and cellular immunity due to mutations in the gene encoding the common gamma (|[gamma]|c) chain. X-SCID is a good candidate for the somatic gene therapy because T and NK cells will develop once the |[gamma]|c chain is introduced into patients' CD34 positive cells in vitro. In addition, recent reports of retrovirus-based X-SCID gene therapy clinical trials from France and England demonstrated the efficacy of the gene therapy. Most of the patients who have received the gene therapy showed NK cell recovery and immunoglobulin production in addition to T cell reconstitution. However, a serious adverse effect was reported in three out of eleven patients treated in France. The complication was lymphoproliferative disease, which occurred about three years later after the gene therapy. Two of them were due to insertional mutagenesis of a proto-oncogene, LMO2 by retroviral vector. Therefore, development of a safer integrating vector is an urgent need. One of the approaches is to incorporate a suicide gene into a therapeutic retrovirus vector to offset the lymphoproliferative disease caused by the gene therapy. In this study, we constructed a retroviral vector encompassing a human |[gamma]|c chain cDNA and a suicide gene from the Herpes simplex virus thymidine kinase (HSVtk). For this purpose, a human |[gamma]|c cDNA was inserted into SFCMM3, a retroviral vector containing an HSVtk gene, which had already been applied in clinical trial. The resultant vector (SFCMM-gamma) was transfected into COS-7 cells and expression of the |[gamma]|c chain was confirmed. For recombinant retrovirus production, the SFCMM-gamma was transfected into an amphotropic packaging cell line, |[psi]|CRIP. 24 hours later after transfection, recombinant virus-producing |[psi]|CRIP cells were irradiated (30Gy) and then co-cultured with EBV transformed B cell lines established from X-SCID patients for 48 hours. The infection cycle was repeated twice. Just prior and a week after starting the co-culture, expression of the |[gamma]|c chain of the EBV-B lines was assessed by flow cytometric analysis. After confirmation of the |[gamma]|c chain expression on the transduced EBV-B lines, the cells were treated with 0.1, 1.0, 10 and 100 mM of ganciclovir (GCV) for 24, 48, 72 and 96 hours. At the end of GCV treatment, expression of the |[gamma]|c chain was analyzed. Cells expressing the transduced |[gamma]|c chain were dramatically reduced within 48 hours after the addition of the GCV, and the |[gamma]|c chain could not be detected on the cell surface after 96 hours with 10 or 100 mM GCV. In contrast, untreated cells expressed the |[gamma]|c chain constantly throughout the experiment. Concentration of GCV up to 10 mM did not affect the growth of the EBV-B cell lines. To test the efficacy and safety feature of the gene therapy in vivo, a murine model of X-SCID was treated dwith the vector. Currently, immunologic reconstitution and efficacy of the suicide gene in this murine model were under investigation.