Long-Term High-Level Reconstitution of NADPH Oxidase Activity in Murine X-Linked Chronic Granulomatous Disease Using a Bicistronic Vector Expressing gp91 phox and a ΔLNGFR Cell Surface Marker

Abstract

A murine model of X-linked chronic granulomatous disease (X-CGD), an inherited immune deficiency with absent phagocyte NADPH oxidase activity caused by defects in the gp91 ^phox gene, was used to evaluate a bicistronic retroviral vector in which expression of human gp91 ^phox and a linked gene for ΔLNGFR, a truncated form of human low-affinity nerve growth factor receptor, are under the control of a spleen focus-forming virus long-terminal repeat (LTR). Four independent cohorts of 11-Gy irradiated X-CGD mice (total, 22 mice) were transplanted with or without preselection of transduced X-CGD bone marrow (BM). Transplanted mice had high-level correction of neutrophil gp91 ^phox expression and reconstitution of NADPH oxidase activity. Expression lasted for at least 14 months in primary transplants, and persisted in secondary and tertiary transplants. Both gp91 ^phox and ΔLNGFR were detected on circulating granulocytes, lymphocytes, lymphoid, and (for ΔLNGFR) red blood cells. Mice receiving transduced bone marrow [BM] preselected ex vivo for ΔLNGFR expression had high-level (= 80%) reconstitution with transduced cells, with an improved fraction of oxidase-corrected neutrophils posttransplant. Analysis of secondary and tertiary CFU-S showed that silencing of individual provirus integrants can occur even after preselection forΔLNGFR prior to transplantation, and that persistent provirus expression was associated with multiple integration sites in most cases. No obvious adverse consequences of transgenic protein expression were observed.