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 lymphohematop oietic system, whereby phagocytes of affected patients are unable to k ill microorganisms. CGD is caused by a functional defect in the phagoc ytic nicotinamide adenine dinucleotide phosphatase (NADPH) oxidase (ph ox) 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), Ful l reconstitution of superoxide-generating activity was achieved with t his vector in a gp91-phox-deficient cell line. Using an optimized gene transfer protocol, up to 85% of the CD34(+) cells obtained from the b one marrow of X-CGD patients were transduced, CD15(+) cells differenti ated 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, immuno blots prepared from extracts off transduced CD15(+) cells revealed gp9 1-phox protein levels similar to those found in neutrophils derived fr om normal CD34(+) cells. Taking into consideration that superoxide pro duction in only 5 to 10% of wild-type neutrophils is sufficient to pro tect 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.