Correction of respiratory burst activity in X-linked chronic granulomatous cells to therapeutically relevant levels after gene transfer into bone marrow CD34+ cells

Chronic granulomatous disease (CGD) is a disorder of the lymphohematopoietic system, whereby phagocytes of affected patients are unable to kill microorganisms. CGD is caused by a functional defect in the phagocytic nicotinamide adenine dinucleotide phosphatase (NADPH) oxidase (phox) enzyme complex, leading to a lack of microbicidal metabolites, As a therapeutic approach toward the predominant X-linked form of CGD, we have developed a bicistronic retroviral vector containing the coding sequences of gp91-phox and a cytoplasmically truncated version of the human low-affinity receptor for nerve growth factor (Delta LNGFR), Full reconstitution of superoxide-generating activity was achieved with this vector in a gp91-phox-deficient cell line. Using an optimized gene transfer protocol, up to 85% of the CD34(+) cells obtained from the bone marrow of X-CGD patients were transduced, CD15(+) cells differentiated in vitro from transduced X-CGD CD34(+) cells showed correction of NADPH oxidase activity to 45-52% of normal levels whereas Delta LNGFR expression was found in 40-67% of the CD15(+) cells. Moreover, immunoblots prepared from extracts off transduced CD15(+) cells revealed gp91-phox protein levels similar to those found in neutrophils derived from normal CD34(+) cells. Taking into consideration that superoxide production in only 5 to 10% of wild-type neutrophils is sufficient to protect X-CGD heterozygotes from serious infections, the results achieved in this study shows that for X-GD patients a curative approach based on the genetic modification of hematopoietic stem/progenitor cells is feasible.