cathepsin-g and Granulomatous-Disease--Chronic

X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene encoding the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase catalytic subunit gp91(phox). A recent clinical trial for X-CGD using a spleen focus-forming virus (SFFV)-based γ-retroviral vector has demonstrated clear therapeutic benefits in several patients although complicated by enhancer-mediated mutagenesis and diminution of effectiveness over time due to silencing of the viral long terminal repeat (LTR). To improve safety and efficacy, we have designed a lentiviral vector that directs transgene expression primarily in myeloid cells. To this end, we created a synthetic chimeric promoter that contains binding sites for myeloid transcription factors CAAT box enhancer-binding family proteins (C/EBPs) and PU.1, which are highly expressed during granulocytic differentiation. As predicted, the chimeric promoter regulated higher reporter gene expression in myeloid than in nonmyeloid cells, and in human hematopoietic progenitors upon granulocytic differentiation. In a murine model of stem cell gene therapy for X-CGD, the chimeric vector resulted in high levels of gp91(phox) expression in committed myeloid cells and granulocytes, and restored normal NADPH-oxidase activity. These findings were recapitulated in human neutrophils derived from transduced X-CGD CD34(+) cells in vivo, and suggest that the chimeric promoter will have utility for gene therapy of myeloid lineage disorders such as CGD.

Topics: Animals; Base Sequence; Binding Sites; Cathepsin G; CCAAT-Enhancer-Binding Proteins; Cell Differentiation; Cell Line, Tumor; DNA Copy Number Variations; Enhancer Elements, Genetic; Gene Expression Regulation; Genes, X-Linked; Genetic Therapy; Genetic Vectors; Granulocytes; Granulomatous Disease, Chronic; Hematopoietic Stem Cells; Humans; Lentivirus; Mice; Molecular Sequence Data; Mutagenesis; Myeloid Cells; NADPH Oxidases; Promoter Regions, Genetic; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fes; Receptors, Immunologic; Recombinant Fusion Proteins; Retroviridae; Spleen Focus-Forming Viruses; Stem Cells; Terminal Repeat Sequences; Trans-Activators; Transgenes

Although several genetic defects are known to impair oxidative microbicidal/cytotoxic mechanisms in human PMN, no deficiencies of PMN granule components that mediate oxygen-independent microbicidal activity have yet been reported. We analyzed PMN from patients with various granulocyte disorders for their content of two azurophil granule constituents, defensins and cathepsin G, that exert microbicidal/cytotoxic activity in vitro, and one component, elastase, that has ancillary microbicidal/cytotoxic activity. PMN from two (of two) patients with specific granule deficiency (SGD) displayed an almost complete deficiency of defensins, which in normal cells constitute greater than 30% of the protein content of azurophil granules. The SGD PMN contained normal or mildly decreased amounts of cathepsin G and elastase. Conversely, the PMN of three (of three) patients with Chediak-Higashi syndrome (CHS) substantially lacked cathepsin G and elastase, but their defensin content was normal or mildly decreased. Both CHS and SGD patients suffer from frequent and severe bacterial infections, and CHS patients frequently develop an atypical lymphoproliferative syndrome. The profound deficiency of PMN components with microbicidal/cytotoxic activity in SGD and CHS may contribute to the clinical manifestations of these disorders.

Topics: Blood Bactericidal Activity; Blood Proteins; Cathepsin G; Cathepsins; Chediak-Higashi Syndrome; Cytoplasmic Granules; Cytotoxins; Defensins; Electrophoresis, Polyacrylamide Gel; Granulomatous Disease, Chronic; Humans; Immunoassay; Neutrophils; Pancreatic Elastase; Peroxidase; Serine Endopeptidases