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 (deltaLNGFR). 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 deltaLNGFR expression was found in 40-67% of the CD15+ cells. Moreover, immunoblots prepared from extracts of 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-CGD patients a curative approach based on the genetic modification of hematopoietic stem/progenitor cells is feasible.
Becker
S
S
Laboratory for Molecular Virology, Georg-Speyer-Haus, Frankfurt, Germany.
Wasser
S
S
Hauses
M
M
Hossle
J P
JP
Ott
M G
MG
Dinauer
M C
MC
Ganser
A
A
Hoelzer
D
D
Seger
R
R
Grez
M
M
D018952:Antigens, CD34 / Q000235:genetics* / Q000378:metabolism; D001854:Bone Marrow Cells / Q000378:metabolism*; D002460:Cell Line; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D008040:Genetic Linkage; D015316:Genetic Therapy; D005822:Genetic Vectors; D006105:Granulomatous Disease, Chronic / Q000235:genetics* / Q000628:therapy*; D006801:Humans; D016256:Lewis X Antigen / Q000276:immunology; D008562:Membrane Glycoproteins / Q000235:genetics*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D009414:Nerve Growth Factors / Q000235:genetics / Q000378:metabolism; D016897:Respiratory Burst; D012190:Retroviridae / Q000235:genetics; D014960:X Chromosome
D018952:Antigens, CD34; D016256:Lewis X Antigen; D008562:Membrane Glycoproteins; D009414:Nerve Growth Factors; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases