Functional Restoration of gp91phox-Oxidase Activity by BAC Transgenesis and Gene Targeting in X-linked Chronic Granulomatous Disease iPSCs

Defects in gp91-phox, the large subunit of cytochrome b558 (b-245) give rise to X-linked chronic granulomatous disease (CGD), a rare inherited condition characterized by an extreme susceptibility to bacterial and fungal infection. In the majority of cases, the phagocytes are unable to generate any superoxide owing to complete absence of the flavocytochrome. However, a small minority of these patients do have some phagocytic oxidase activity. We describe here an analysis of the molecular basis of the disease in three such variant patients with lesions in the gene coding for gp91-phox on the X chromosome. Three different genetic lesions were found, resulting in the substitution of tyrosine for cysteine 244, a deletion of one of three lysines 313 through 315, and the deletion of the six C-terminal amino acids, respectively. The functional consequences of these defects on oxidase activity was a reduction to 12%, 3.6%, and 2.1% of the normal levels, respectively. Corresponding levels of gp91-phox were 20%, 8%, and 16% of normal classifying these patients as X91-. Microbicidal assays showed that killing of Staphylococcus aureus was grossly impaired in cells in which there was 12% normal activity. This implies that if gene therapy is to be applied, it must restore oxidase activity to a much higher level than that present in the cells of this patient. The sites of two of the mutations were analyzed on a model of the C-terminal half of the gp91-phox, based on the crystal structure of the homologous protein ferrodoxin NADP reductase. Possible structural consequences of the mutations were examined.

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NADPH oxidase correction by mRNA transfection of apheresis granulocytes in chronic granulomatous disease

Blood Advances ◽

10.1182/bloodadvances.2020003224 ◽

2020 ◽

Vol 4 (23) ◽

pp. 5976-5987

Author(s):

Suk See De Ravin ◽

Julie Brault ◽

Ronald J. Meis ◽

Linhong Li ◽

Narda Theobald ◽

...

Keyword(s):

Nadph Oxidase ◽

Chronic Granulomatous Disease ◽

Messenger Rna ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Functional Restoration ◽

In Vivo Studies ◽

Allogeneic Bone ◽

Granulomatous Disease ◽

Primate Model

Abstract Granulocytes from patients with chronic granulomatous disease (CGD) have dysfunctional phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase that fails to generate sufficient antimicrobial reactive oxidative species. CGD patients with severe persistent fungal or bacterial infection who do not respond to antibiotic therapy may be given apheresis-derived allogeneic granulocyte transfusions from healthy volunteers to improve clearance of intractable infections. Allogeneic granulocyte donors are not HLA matched, so patients who receive the donor granulocyte products may develop anti-HLA alloimmunity. This not only precludes future use of allogeneic granulocytes in an alloimmunized CGD recipient, but increases the risk of graft failure of those recipients who go on to need an allogeneic bone marrow transplant. Here, we provide the first demonstration of efficient functional restoration of CGD patient apheresis granulocytes by messenger RNA (mRNA) electroporation using a scalable, Good Manufacturing Practice–compliant system to restore protein expression and NADPH oxidase function. Dose-escalating clinical-scale in vivo studies in a nonhuman primate model verify the feasibility, safety, and persistence in peripheral blood of infusions of mRNA-transfected autologous granulocyte-enriched apheresis cells, supporting this novel therapeutic approach as a potential nonalloimmunizing adjunct treatment of intractable infections in CGD patients.

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Deficiency of NADPH oxidase activity in chronic granulomatous disease

The Journal of Pediatrics ◽

10.1016/s0022-3476(77)80632-x ◽

1977 ◽

Vol 90 (2) ◽

pp. 213-217 ◽

Cited By ~ 37

Author(s):

Linda C. McPhail ◽

Lawrence R. DeChatelet ◽

Pamela S. Shirley ◽

Catherine Wilfert ◽

Richard B. Johnston ◽

...

Keyword(s):

Nadph Oxidase ◽

Chronic Granulomatous Disease ◽

Oxidase Activity ◽

Granulomatous Disease ◽

Nadph Oxidase Activity

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A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40phox and selective defects in neutrophil NADPH oxidase activity

Blood ◽

10.1182/blood-2009-07-231498 ◽

2009 ◽

Vol 114 (15) ◽

pp. 3309-3315 ◽

Cited By ~ 287

Author(s):

Juan D. Matute ◽

Andres A. Arias ◽

Nicola A. M. Wright ◽

Iwona Wrobel ◽

Christopher C. M. Waterhouse ◽

...

Keyword(s):

Nadph Oxidase ◽

Chronic Granulomatous Disease ◽

Oxidase Activity ◽

Autosomal Recessive ◽

Premature Stop Codon ◽

Superoxide Production ◽

Human Neutrophils ◽

Granulomatous Disease ◽

Recessive Mutations ◽

Px Domain

Abstract Chronic granulomatous disease (CGD), an immunodeficiency with recurrent pyogenic infections and granulomatous inflammation, results from loss of phagocyte superoxide production by recessive mutations in any 1 of 4 genes encoding subunits of the phagocyte NADPH oxidase. These include gp91phox and p22phox, which form the membrane-integrated flavocytochrome b, and cytosolic subunits p47phox and p67phox. A fifth subunit, p40phox, plays an important role in phagocytosis-induced superoxide production via a phox homology (PX) domain that binds to phosphatidylinositol 3-phosphate (PtdIns(3)P). We report the first case of autosomal recessive mutations in NCF4, the gene encoding p40phox, in a boy who presented with granulomatous colitis. His neutrophils showed a substantial defect in intracellular superoxide production during phagocytosis, whereas extracellular release of superoxide elicited by phorbol ester or formyl-methionyl-leucyl-phenylalanine (fMLF) was unaffected. Genetic analysis of NCF4 showed compound heterozygosity for a frameshift mutation with premature stop codon and a missense mutation predicting a R105Q substitution in the PX domain. Parents and a sibling were healthy heterozygous carriers. p40phoxR105Q lacked binding to PtdIns(3)P and failed to reconstitute phagocytosis-induced oxidase activity in p40phox-deficient granulocytes, with premature loss of p40phoxR105Q from phagosomes. Thus, p40phox binding to PtdIns(3)P is essential for phagocytosis-induced oxidant production in human neutrophils and its absence can be associated with disease.

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Late-Onset of Chronic Granulomatous Disease Revealed By Paecilomyces Lilacinus Cutaneous Infection

10.21203/rs.3.rs-516219/v1 ◽

2021 ◽

Author(s):

Clément Lemaigre ◽

Felipe Suarez ◽

Jean-Philippe Martellosio ◽

Cindy Barbarin ◽

Kevin Brunet ◽

...

Keyword(s):

Nadph Oxidase ◽

Chronic Granulomatous Disease ◽

Oxidase Activity ◽

Late Onset ◽

Asymptomatic Carrier ◽

Clinical Manifestations ◽

Female Carrier ◽

Granulomatous Disease ◽

Nadph Oxidase Activity ◽

Inherited Immunodeficiency

Abstract Chronic granulomatous disease (CGD) is an inherited immunodeficiency due to defective leukocyte NADPH responsible for recurrent infections and aberrant inflammation. Mutations in the CYBB gene are responsible for the X-linked CGD and account for approximately 70% of the cases. CGD is diagnosed during childhood in males. Female carriers may have biased X inactivation and may present with clinical manifestations depending on the level of residual NADPH oxidase activity. We report the case of a previously asymptomatic female carrier who was diagnosed at age 67 with a skin infection with the rare fungus, Paecylomyces lilacinus as the first manifestation of CGD. Dihydrorhodamin 123 (DHR) activity was below 10%. Next-generation sequencing (NGS) revealed mutations in DNMT3A, ASXL1, and STAG2 suggesting that clonal hematopoiesis of undetermined potential (CHIP) could be responsible for a progressive loss of NADPH oxidase activity and the late onset of X-linked CGD in this patient. Long-term follow-up of asymptomatic carrier women seems to be essential after 50 years old.

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Prolonged recombinant interferon-gamma therapy in chronic granulomatous disease: evidence against enhanced neutrophil oxidase activity

Blood ◽

10.1182/blood.v79.6.1558.bloodjournal7961558 ◽

1992 ◽

Vol 79 (6) ◽

pp. 1558-1562 ◽

Cited By ~ 1

Author(s):

RC Woodman ◽

RW Erickson ◽

J Rae ◽

HS Jaffe ◽

JT Curnutte

Keyword(s):

Nadph Oxidase ◽

Chronic Granulomatous Disease ◽

Interferon Gamma ◽

Oxidase Activity ◽

Small Subset ◽

Variant Form ◽

Granulomatous Disease ◽

Recombinant Interferon ◽

Nadph Oxidase Activity ◽

O2 Production

Recombinant interferon-gamma (rIFN-gamma) therapy has become an effective form of prophylaxis for patients with chronic granulomatous disease (CGD). Preliminary studies with CGD suggested that rIFN-gamma treatment enhanced phagocyte oxidase activity and increased superoxide (O2-) production. We evaluated several aspects of neutrophil NADPH oxidase activity in 19 CGD patients (representing all four known types of CGD) receiving prolonged rIFN-gamma therapy (6 to 27 months). In contrast to earlier studies, we failed to detect any improvement in neutrophil NADPH oxidase activity in 18 of the 19 CGD patients as determined by (1) intact cell O2- production (continuous assay), (2) nitroblue tetrazolium (NBT) staining, (3) cytochrome b558 spectroscopy, and (4) activity levels of cytosol and membrane oxidase components using a cell-free activation system. One patient with a variant form of X-linked CGD had a transient increase in neutrophil O2- production following 3 months of rIFN-gamma therapy. However, this was not sustained, and was not associated with any change in cytochrome b levels. In some patients, rIFN-gamma therapy was associated with the appearance of a small subset of circulating monocytes (1% to 20%) that were NBT-positive. Although the functional significance of this monocyte subpopulation needs to be determined, these results suggest that one possible mechanism by which rIFN-gamma may benefit CGD patients is by partially correcting the respiratory burst defect in a subset of monocytes. We conclude that the clinical benefit of prolonged rIFN-gamma therapy in the vast majority of CGD patients is not due to enhanced neutrophil NADPH oxidase activity. The mechanism of action of rIFN-gamma in most CGD patients remains unknown.

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Heightened turnover and failed maturation of monocyte-derived macrophages in murine Chronic Granulomatous Disease

Blood ◽

10.1182/blood.2021011798 ◽

2021 ◽

Author(s):

Sophie Louise Gibbings ◽

Kelsey Haist ◽

Heidi J Nick ◽

S Courtney Frasch ◽

Teagan H Glass ◽

...

Keyword(s):

Bone Marrow ◽

Chronic Granulomatous Disease ◽

Adoptive Transfer ◽

Oxidase Activity ◽

Granulomatous Disease ◽

Wild Type ◽

Nadph Oxidase Activity ◽

Normal Behavior ◽

Bone Marrow Chimeras ◽

Over Time

Loss of NADPH oxidase activity leads to altered phagocyte responses and exaggerated inflammation in Chronic Granulomatous Disease (CGD). We sought to assess the effects of Nox2 absence on monocyte-derived macrophages (MoMacs) in gp91phox-/y mice during zymosan-induced peritonitis. MoMacs from CGD and wild type (WT) peritonea were characterized over time after zymosan injection. Though numbers lavaged from both genotypes were virtually identical, there were marked differences in maturation: newly recruited WT MoMacs rapidly enlarged and matured, losing Ly6C and gaining MHCII, CD206 and CD36, while CGD MoMacs remained small and were mostly Ly6C+MHCII-. RNAseq analyses showed few intrinsic differences between genotypes in newly recruited MoMacs but significant differences with time. WT MoMacs demonstrated changes in metabolism, adhesion and reparative functions, while CGD MoMacs remained inflammatory. PKH dye labeling demonstrated that while WT MoMacs were mostly recruited within the first 24h and remained in the peritoneum while maturing and enlarging, CGD monocytes streamed into the peritoneum for days with many migrating to the diaphragm where they were found in fibrin(ogen) clots surrounding clusters of neutrophils in nascent pyogranulomata. Importantly, these observations appeared to be driven by milieu: adoptive transfer of CGD MoMacs into inflamed peritonea of WT mice resulted in immunophenotypic maturation and normal behavior, whereas altered maturation/behavior of WT MoMacs resulted from transfer into inflamed peritonea of CGD mice. Additionally, Nox2-deficient MoMacs behaved similarly to their Nox2-sufficient counterparts within the largely WT milieu of mixed bone marrow chimeras. These data demonstrate persistent recruitment with fundamental failure of MoMac maturation in CGD.

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Gene Therapy with pCCLchimP47 -phox Lentiviral Vector Achieves Long-term Reconstitution of NADPH Oxidase Activity in p47 -phox Chronic Granulomatous Disease (CGD) Mouse Model

Journal of Allergy and Clinical Immunology ◽

10.1016/j.jaci.2017.12.623 ◽

2018 ◽

Vol 141 (2) ◽

pp. AB197

Author(s):

Walmir Cutrim Aragao Filho ◽

Andrea Shejtman ◽

Marta Zinicola ◽

Ulrich Siler ◽

Janine Reichenbach ◽

...

Keyword(s):

Gene Therapy ◽

Mouse Model ◽

Nadph Oxidase ◽

Chronic Granulomatous Disease ◽

Oxidase Activity ◽

Lentiviral Vector ◽

Granulomatous Disease ◽

Nadph Oxidase Activity

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Retrovirus gene therapy for X-linked chronic granulomatous disease can achieve stable long-term correction of oxidase activity in peripheral blood neutrophils

Blood ◽

10.1182/blood-2009-05-222760 ◽

2010 ◽

Vol 115 (4) ◽

pp. 783-791 ◽

Cited By ~ 135

Author(s):

Elizabeth M. Kang ◽

Uimook Choi ◽

Narda Theobald ◽

Gilda Linton ◽

Debra A. Long Priel ◽

...

Keyword(s):

Gene Therapy ◽

Chronic Granulomatous Disease ◽

Oxidase Activity ◽

Severe Infection ◽

Granulomatous Disease ◽

Gene Therapy Trial ◽

Blood Neutrophils ◽

Severe Infections ◽

Therapy Trial

Abstract Chronic granulomatous disease (CGD) is associated with significant morbidity and mortality from infection. The first CGD gene therapy trial resulted in only short-term marking of 0.01% to 0.1% of neutrophils. A recent study, using busulfan conditioning and an SFFV retrovirus vector, achieved more than 20% marking in 2 patients with X-linked CGD. However, oxidase correction per marked neutrophil was less than normal and not sustained. Despite this, patients clearly benefited in that severe infections resolved. As such, we initiated a gene therapy trial for X-CGD to treat severe infections unresponsive to conventional therapy. We treated 3 adult patients using busulfan conditioning and an MFGS retroviral vector encoding gp91phox, achieving early marking of 26%, 5%, and 4% of neutrophils, respectively, with sustained long-term marking of 1.1% and 0.03% of neutrophils in 2 of the patients. Gene-marked neutrophils have sustained full correction of oxidase activity for 34 and 11 months, respectively, with full or partial resolution of infection in those 2 patients. Gene marking is polyclonal with no clonal dominance. We conclude that busulfan conditioning together with an MFGS vector is capable of achieving long-term correction of neutrophil oxidase function sufficient to provide benefit in management of severe infection. This study was registered at www.clinicaltrials.gov as #NCT00394316.

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