title: Signed outside: a surface marker system for transgenic cytoplasmic proteins.
abstract: Chronic granulomatous disease is a primary immunodeficiency, comprising five molecular defects, characterized by an impaired respiratory burst activity of myeloid cells. We are currently developing a gene therapy vector for the p47phox-deficient form of chronic granulomatous disease. Classic intracellular immunostaining of the cytoplasmic p47phox transgene product, however, interferes with respiratory burst activity. In this study we report a new system for measuring p47phox expression: A single open reading frame encoding the surface marker protein ΔLNGFR (truncated low-affinity nerve growth factor receptor) linked to the p47phox transgene by the 2A oligopeptide coexpression technology. Translation generates two discrete products: p47phox localizing to the cytoplasm and 'ΔLNGFR-2A' localizing to the cell surface. Six weeks after transplantation of transduced autologous hematopoietic stem cells into p47-/- mice, the intracellular p47phox fluorescence-activated cell sorting (FACS) signal intensities corresponded to surface ΔLNGFR staining in monocytes, B cells, T cells and Sca I+ bone marrow cells in vivo. The p47phox cleavage product restored nicotinamide adenine dinucleotide phosphate-oxidase activity in granulocytes differentiated from transduced p47phox-/- murine hematopoietic stem cells ex vivo, in murine granulocytes/monocytes in vivo, and in transduced human monocyte derived macrophages from p47phox-deficient chronic granulomatous disease patients. In conclusion, this new marker system allows highly efficient, indirect detection of cytoplasmic transgene products by FACS surface staining.
authors.affiliation: Division of Immunology/Hematology/BMT, University Children's Hospital Zürich, Steinwiesstrasse 75, Zürich, Switzerland.
authors.forename: V
authors.initials: V
authors.lastname: Wohlgensinger
authors.forename: R
authors.initials: R
authors.lastname: Seger
authors.forename: M D
authors.initials: MD
authors.lastname: Ryan
authors.forename: J
authors.initials: J
authors.lastname: Reichenbach
authors.forename: U
authors.initials: U
authors.lastname: Siler
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000737:chemistry; D005434:Flow Cytometry; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D006105:Granulomatous Disease, Chronic / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D051379:Mice; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism; D017475:Receptors, Nerve Growth Factor / Q000235:genetics*; D019076:Transgenes / Q000235:genetics*
chemical_list: D015415:Biomarkers; D017475:Receptors, Nerve Growth Factor; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1