Gene therapy of chronic granulomatous disease with GFP-tagged retrovirus vectors

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To improve gene therapy vectors for X-linked chronic granulomatous disease (X-CGD), retroviral cis-elements were investigated for efficient gene transfer and expression. The vectors were evaluated for exprssion levels of the therapeutic gp91 gene and the green fluorescent protein (GFP) marker gene. We found the long terminal repeats and the primer binding site from MSCV were efficient in gene transfer and long-term expression, and the splicing signals from MFG were beneficial for strong transgene expression. Therefore we hooked up these cis-elements from MSCV and MFG to construct a new retroviral backbone, MGK.An MGK-derived bicistronic vector gave efficient gene transfer into X-CGD bone marrow cells and good transgene expression, which corrected the CGD phenotype in the treated animals. We have also investigated the feasibility of in vivo expansion of gene-modified hematopoietic cells. For this purpose, we have developed "selective amplifier genes", which encode fusion proteins between the granulocyte colony-stimulating factor receptor (GCSFR) and the ligand-binding domains (LBDs) of steroid receptors. The LBD, in our case specifically binds to 4-hydroxytamoxifen (4-HT), functions as a molecular switch to convert GCSFR into a ligand-dependent growth signal generator. After reconstituting murine hematopoiesis with the bone marrow cells transduced by a bicistronic retrovirus containing the selective amplifier gene and the GFP gene, the recipients were challenged with 4-HT.The challenged mice had significantly greater proportion of GFP+ leukocytes than the controls. The expanded cells contained more granulocytes/monocytes than lymphocytes, the target lineage cells of CGD gene therapy. We are planning to construct MGK-based bicistronic retroviral vectors containing the gp91 gene and the selective amplifier gene, for preclinical studies with X-CGD mice.