Chronic Granulomatous Disease Patients Research Articles

A nox2/cybb zebrafish mutant with defective myeloid cell reactive oxygen species production displays normal initial neutrophil recruitment to sterile tail injuries.

Reactive oxygen species are important effectors and modifiers of the acute inflammatory response, recruiting phagocytes including neutrophils to sites of tissue injury. In turn, phagocytes such as neutrophils are both consumers and producers of reactive oxygen species. Phagocytes including neutrophils generate reactive oxygen species in an oxidative burst through the activity of a multimeric phagocytic nicotinamide adenine dinucleotide phosphate oxidase complex. Mutations in the NOX2/CYBB (previously gp91phox) nicotinamide adenine dinucleotide phosphate oxidase subunit are the commonest cause of chronic granulomatous disease, a disease characterized by infection susceptibility and an inflammatory phenotype. To model chronic granulomatous disease, we made a nox2/cybb zebrafish (Danio rerio) mutant and demonstrated it to have severely impaired myeloid cell reactive oxygen species production. Reduced early survival of nox2 mutant embryos indicated an essential requirement for nox2 during early development. In nox2/cybb zebrafish mutants, the dynamics of initial neutrophil recruitment to both mild and severe surgical tailfin wounds was normal, suggesting that excessive neutrophil recruitment at the initiation of inflammation is not the primary cause of the "sterile" inflammatory phenotype of chronic granulomatous disease patients. This nox2 zebrafish mutant adds to existing in vivo models for studying reactive oxygen species function in myeloid cells including neutrophils in development and disease.

Inflammatory Complications in Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is a rare inborn error of immunity that typically manifests with infectious complications. As the name suggest though, inflammatory complications are also common, often affecting the gastrointestinal, respiratory, urinary tracts and other tissues. These can be seen in all various types of CGD, from X-linked and autosomal recessive to X-linked carriers. The pathogenetic mechanisms underlying these complications are not well understood, but are likely multi-factorial and reflect the body's attempt to control infections. The different levels of neutrophil residual oxidase activity are thought to contribute to the large phenotypic variations. Immunosuppressive agents have traditionally been used to treat these complications, but their use is hindered by the fact that CGD patients are predisposed to infection. Novel therapeutic agents, like anti-TNFa monoclonal antibodies, anakinra, ustekinumab, and vedolizumab offer promise for the future, while hematopoietic stem cell transplantation should also be considered in these patients.

Generation of a human iPSC line (CHCMUi002-A) from peripheral blood mononuclear cells from a chronic granulomatous disease patient

Chronic granulomatous disease (CGD) is a rare X-linked recessive primary immunodeficiency disease (PID). Herein, a human induced pluripotent stem cell (iPSC) line was generated from the peripheral blood mononuclear cells (PBMCs) of a CGD patient with a mutation (c.785_786delTT) in the CYBB gene. These iPSCs showed the expression of pluripotency markers, the ability to differentiate into three germ layers. They offer a promising technique for studying the pathogenesis and conducting drug screening for CGD patients.

Prime Editing Efficiently and Precisely Corrects Causative Mutation in Chronic Granulomatous Disease, Restoring Myeloid Function: Toward Development of a Prime Edited Autologous Hematopoietic Stem Cell Therapy

Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency characterized by a lack of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in phagocytic myeloid cells such as neutrophils. NADPH oxidase produces reactive oxygen species (ROS) that directly kill bacteria and fungi to control infection. CGD patients lack this ability to control infection, which results in chronic and severe infections, inflammation, autoimmunity, poor quality of life, and reduced lifespan. Current standard care for CGD is antimicrobial prophylaxis and aggressive management of infection. For patients with a suitable human leukocyte antigen (HLA) matched donor, an allogeneic hematopoietic stem cell (HSC) transplant (allo-HSCT) offers a curative approach. However, many patients do not have access to HLA matched donors and allo-HSCT also carries a risk of GvHD and graft rejection. P47phox-deficient CGD, the most common autosomal recessive CGD, is caused by mutations in the NCF1 gene, which encodes the p47phox protein, a subunit of the NADPH oxidase complex. Approximately 80% of p47phox-deficient CGD patients carry the same homozygous mutation, a 2 nucleotide GT deletion (delGT) in exon 2 of NCF1 which is correctable using Prime Editing. Transplantation of Prime Edited patient autologous CD34 + cells, in which the delGT mutation has been corrected with Prime Editing, may provide a curative treatment for p47phox-deficient CGD patients. To evaluate whether correction of the delGT NCF1 mutation in CGD patient CD34 + cells restores p47phox protein expression and NADPH oxidase activity in myeloid progeny, Prime Editors were designed to correct this mutation precisely and efficiently. The Prime Editors were delivered to CD34 + cells from two p47phox-deficient CGD patients homozygous for delGT NCF1. In both studies, ≧75% of Prime Edited patient CD34 + cells carried at least 1 corrected allele as determined by ddPCR and DNA sequencing analyses. Myeloid progeny differentiated from Prime Edited patient CD34 + cells had restored p47phox protein expression and NADPH oxidase activity to ~80% of normal healthy donors as determined by multiple ROS detection assays. To evaluate reproducibility, Prime Editors were delivered to CD34 + cells from 6 different healthy donors. On average, greater than 90% of CD34 + cells were Prime Edited across 6 donors in independent experiments. To confirm that Prime Edited CD34 + cells retain stem cell multipotency, engraftment, and hematopoietic reconstitution properties, Prime Edited and mock treated CD34 + cells from three different donors were transplanted into immunodeficient mice. Sixteen weeks after CD34 + cell transplantation, >87% of long-term hematopoietic stem cells (LT-HSC) that repopulated the bone marrow had at least 1 allele corrected. There were no significant differences between ‘electroporation only’ mock treated and Prime Editor electroporated human CD34 + cells with respect to long-term CD34 + cell engraftment, human CD45 + blood cell chimerism, hematopoietic multilineage blood cell reconstitution and biodistribution, or LT-HSC cell potency. Across these studies, >95% human CD45 + cell chimerism was achieved with Prime Edited CD34 + cells. Despite high on-target correction of the delGT mutation in NCF1, off-target editing, unintended editing, and chromosomal rearrangements (larger deletions or translocation) events were not detected using a robust suite of genome-wide assays. In summary, Prime Editing corrects a mutation that causes the most common autosomal recessive from of CGD; correction of this delGT mutation at NCF1 restores NADPH oxidase activity and function in myeloid progeny of Prime Edited CGD CD34 + cells; and Prime Edited long-term HSCs retain multipotency and functionality in vivo. These results support the development of a Prime Edited CGD patient autologous CD34 + cell therapy as a potential curative approach for p47phox-deficient CGD patients.

NOX2 deficiency enhances priming and activation of the NLRP3 inflammasome

Nicotinamide adenine dinucleotide phosphate oxidase complex 2 (NOX2) deficiency, or chronic granulomatous disease (CGD), is an inborn error of immunity associated with increased susceptibility to infection and inflammatory manifestations. The pathophysiologic mechanism leading to the increased inflammatory response in CGD remains elusive. We investigated the pathophysiologic mechanisms leading to NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation in NOX2 deficiency. We used NOX2-deficient human primary and CRISPR-engineered macrophages to show that NOX2 deficiency enhances the inflammatory response mainly by modulating the 2 steps of NLRP3 inflammasome activation: its transcriptional priming and its posttranslational triggering. At the transcriptional level, NOX2-deficient phagocytes display increased priming of the NLRP3 inflammasome, as evidenced by increased transcription of NLRP3 and IL-1β through an IL-1β-dependent stimulation of the nuclear factor kappa-light-chain enhancer of activated B cells (aka NF-κB) pathway. At the posttranslational level, the absence of NOX2 triggers the NLRP3 inflammasome activation by increased K+ efflux and excessive release of mitochondrial DNA due to mitochondrial damage. Furthermore, NLRP3-driven pyroptosis in NOX2-deficient phagocytes further enhances NLRP3 activation by increasing K+ efflux. Our results unveil the role of NOX2 as a repressor of the inflammatory response at both transcriptional and posttranslational levels and pave the way for a more targeted approach to treating CGD patients with inflammatory manifestations.

Carrier State of Chronic Granulomatous Disease Presented at Middle Age: A Case Report

Introduction: Chronic granulomatous disease (CGD) is the most common inherited defect of phagocytes. Although most female carriers of X-linked CGD have been considered to be unaffected, they may have similar problems to those of CGD patients. This study suggests that the CGD carrier state might be more complex than it was previously appreciated. Case Presentation: A sixty-year-old woman visited our hospital in June 2021 due to pneumonia and pleural effusion. Chest computed tomography scan revealed left lower lobe pneumonia and mediastinal lymphadenopathies. In July 2021, her symptoms (high-grade fever, chills, and hemoptysis) were initially attributed to a hydatid cyst. Therefore, she underwent a lobectomy, resulting in purulent drainage on the excision site on the skin, refractory to local and systemic treatments. The refractory and recurrent nature of the lesions led to the immunological evaluation, which was completely normal with a 100% nitroblue tetrazolium (NBT) and a dihydrorhodamine (DHR) of 278 (normal > 100). Further DHR123 flow cytometry investigations with multiple stimulants revealed a carrier state of CGD, which was relevant to her history of chronic complications. Conclusions: Adult patients with unusual manifestations suggestive of neutrophil function defects in adulthood should be evaluated for the CGD carrier state. In these cases, NBT alone may miss the diagnosis of CGD. In such cases, DHR testing with multiple stimulants may establish a robust diagnosis.

Peripheral T Cell Populations are Differentially Affected in Familial Mediterranean Fever, Chronic Granulomatous Disease, and Gout

Both innate errors of immunity, such as familial Mediterranean fever (FMF) and chronic granulomatous disease (CGD), and the common inflammatory disease gout are characterized by episodes of sterile inflammatory attacks in the absence of an infection. While these disorders encompass distinct pathologies due to differentially affected metabolic pathways and inflammasome activation mechanisms, their common features are the excessive production of interleukin (IL)-1ß and innate immune cell hyperreactivity. On the other hand, the role of T cells and innate-like lymphocytes such as gamma delta (γδ) T cells in these pathologies is ill-defined. In order to widen our understanding of T cell involvement in CGD, FMF and gout pathology, we developed multicolour immunophenotyping panels for flow cytometry to characterize γδ T cells as well as CD4 and CD8 T cell populations in terms of their cytokine production, activation status, memory or naive phenotypes, exhaustion status, homing receptor expression, and cytotoxic activity. Our study is the first deep immunophenotyping analysis of T cell populations in CGD, FMF, and gout patients. We found that CGD affects the frequencies and activation status of T cells, while gout impairs the cytokine production capacity of Vδ2 T cells. FMF was characterized by decreased percentages of regulatory T cells in circulation and attenuated IFN-γ production capacity by Vδ2 T cells. Autoinflammatory syndromes and congenital defects of phagocyte differentially affect T cell compartments. Future studies are warranted to assess whether these phenotypical changes are relevant for disease pathology.

Infections due to Salmonella sp. in children with chronic granulomatous disease: Our experience from North India

Infections with non-typhoidal Salmonella sp. have been documented in children with chronic granulomatous disease (CGD), but the prevalence of salmonella infection in children with CGD in underdeveloped countries is unknown. We assessed the clinical profiles of CGD patients diagnosed at our tertiary care centre in north India and had Salmonella sp.infections. We found three patients with Salmonella sp. bloodstream infections (2-proven, 1-probable) among the 99 CGD patients. After receiving cotrimoxazole prophylaxis following a CGD diagnosis, we noted that none of our patients experienced non-typhoidal salmonella infection. One patient experienced severe typhoidal bacteremia despite receipt of cotrimoxazole prophylaxis. This patient required numerous hospital admissions and prolonged intravenous antibiotic regimen. We suggest that vaccination with killed typhoidal vaccines should be regularly given to children with CGD in order to avoid typhoidal bacteremia, in addition to cotrimoxazole prophylaxis and a focus on good hand and food hygiene.

Intestinal microbiome and metabolome signatures in patients with chronic granulomatous disease

Chronic granulomatous disease (CGD) is caused by defects in any 1 of the 6 subunits forming the nicotinamide adenine dinucleotide phosphate oxidase complex 2 (NOX2), leading to severely reduced or absent phagocyte-derived reactive oxygen species production. Almost 50% of patients with CGD have inflammatory bowel disease (CGD-IBD). While conventional IBD therapies can treat CGD-IBD, their benefits must be weighed against the risk of infection. Understanding the impact of NOX2 defects on the intestinal microbiota may lead to the identification of novel CGD-IBD treatments. We sought to identify microbiome and metabolome signatures that can distinguish individuals with CGD and CGD-IBD. We conducted a cross-sectional observational study of 79 patients with CGD, 8 pathogenic variant carriers, and 19 healthy controls followed at the National Institutes of Health Clinical Center. We profiled the intestinal microbiome (amplicon sequencing) and stool metabolome, and validated our findings in a second cohort of 36 patients with CGD recruited through the Primary Immune Deficiency Treatment Consortium. We identified distinct intestinal microbiome and metabolome profiles in patients with CGD compared to healthy individuals. We observed enrichment for Erysipelatoclostridium spp, Sellimonas spp, and Lachnoclostridium spp in CGD stool samples. Despite differences in bacterial alpha and beta diversity between the 2 cohorts, several taxa correlated significantly between both cohorts. We further demonstrated that patients with CGD-IBD have a distinct microbiome and metabolome profile compared to patients without CGD-IBD. Intestinal microbiome and metabolome signatures distinguished patients with CGD and CGD-IBD, and identified potential biomarkers and therapeutic targets.

Spontaneous NETosis and type I IFN signaling activation in resting neutrophils of chronic granulomatous disease patients with CYBB mutations

Spontaneous NETosis and type I IFN signaling activation in resting neutrophils of chronic granulomatous disease patients with CYBB mutations

Inborn Errors of Immunity-the Sri Lankan Experience 2010-2022.

Inborn errors of immunity (IEI) are typically monogenic. Data from the Indian subcontinent are relatively scarce. This paper evaluates IEI diagnosed in Sri Lanka. Data of patients diagnosed with IEI from 2010 to 2022 at the Department of Immunology, Medical Research Institute, Colombo, Sri Lanka, were retrospectively analyzed. Two hundred and six patients were diagnosed with IEI, with a prevalence of 0.94per 100,000. The onset of disease was below 12years in 84.9%, whereas in 10.9%, it was after 18years. The male: female ratio was 1.78:1. Consanguinity was identified in 26.6%. IEI were found in all but one (bone marrow failure) of the 10 IUIS categories. Predominantly antibody deficiencies were the most common category among the nine identified (30.1%), followed by combined immune deficiencies with syndromic features (21.3%), immunodeficiencies affecting cellular and humoral immunity (19.9%), congenital defects of phagocyte number or function (13.1%), and defects in intrinsic and innate immunity (8.2%). Severe combined immune deficiency (SCID) was the commonest disease (14.6%), followed by chronic granulomatous disease (CGD) (10.6%) and X linked agammaglobulinemia (8.7%). Of the patients with a known outcome (n = 184), 51 died (27.7%). Mortality rates were high in SCID (83.3%), Omenn syndrome (OS) (100%), and CGD (31.8%) patients. IEI in Sri Lanka are diagnosed mainly in childhood. The low diagnosis rates suggest a need for educating clinicians regarding IEI in adulthood. The high mortality rates associated with some IEI indicate the need of transplant services in the country.

Chronic Granulomatous Disease: a Cohort of 173 Patients—10-Years Single Center Experience from Egypt

PurposeChronic granulomatous disease (CGD) is an inherited primary immunodeficiency disorder of phagocytes, characterized by recurrent fungal and bacterial infections. Our aim is to describe the different clinical presentations, non-infectious auto-inflammatory features, types and sites of infections, and to estimate the mortality among our large cohort.MethodsThis is a retrospective study conducted at the Pediatric Department of Cairo University Children’s Hospital in Egypt, including cases with a confirmed CGD diagnosis.ResultsOne hundred seventy-three confirmed CGD patients were included. AR-CGD was diagnosed in 132 patients (76.3%) including 83 patients (48%) with p47phox defect, 44 patients (25.4%) with p22phox defect, and 5 patients (2.9%) with p67phox defect. XL-CGD was diagnosed in 25 patients (14.4%). The most common recorded clinical manifestations were deep-seated abscesses and pneumonia. Gram-negative bacteria and Aspergillus were the most frequently isolated species. Regarding the outcome, 36 patients (20.8%) were lost from follow-up. Among patients with known outcome, 94/137 patients (68.6%) are living, while 43/137 patients (31.4%) died.ConclusionAR-CGD is predominant in Egypt; CGD must always be ruled out in any patient presenting with typical or atypical mycobacterial or BCG-disease.

An Unusual Presentation of Pulmonary Mucormycosis as a “Mass-like” Consolidation in an Infant with Chronic Granulomatous: A Case Report Study

Introduction: Chronic granulomatous disease (CGD) is a primary hereditary immunodeficiency condition caused by a defect in the nicotinamide adenine dinucleotide phosphate (NADP) oxidase complex. Recurrent infections and inflammatory complications with a narrow bacterial and fungal spectrum characterize CGD. Acute inflammatory and infectious complications are vexing and fatal for them. Therefore, managing CGD with prophylactic antibiotics and antifungal agents is essential. Case Presentation: We present a 23-month-old infant with a chief complaint of recent lymphadenopathy diagnosed with CGD. Two months later, she was hospitalized with uncontrolled fever and lung involvement while receiving prophylactic treatment with trimethoprim/sulfamethoxazole. A mass-like consolidation was seen in her radiography evaluation. Based on the biopsy and bronchoalveolar lavage (BAL) pathology report, pulmonary mucormycosis was confirmed. Two weeks after starting the standard treatment regimen with amphotericin B, the patient’s symptoms improved. She was discharged in good general condition with oral voriconazole and trimethoprim/sulfamethoxazole and referred to do hematopoietic stem cell transplantation (HSCT). Conclusions: Mucormycosis in CGD is not a common infection and usually infects patients with impaired cell-mediated immunity, hematological malignancies, steroid treatment, and diabetes. Patients with CGD are vulnerable when treated with a high dose of immunosuppressant medications. In our case report, the patient was infected with mucormycosis despite not receiving immunosuppressant medications and having no history of hematologic malignancies or diabetes. Our patient also had a mass-like consolidation in her radiological result, a rare feature of pulmonary mucormycosis involvement in CGD patients.

Characterization of AR-CGD female patient with a novel homozygous deletion in CYBC1 gene presenting with unusual clinical phenotype

Chronic granulomatous disease (CGD) is a human IEI caused by mutations in genes encoding the NADPH oxidase subunits, the enzyme responsible for the respiratory burst. CGD patients have severe life-threatening infections, hyperinflammation and immune dysregulation. Recently, an additional autosomal recessive AR-CGD (type 5) caused by mutations in CYBC1/EROS gene was identified.We report a AR-CGD5 patient with a novel loss of function (LOF) homozygous deletion c.8_7del in the CYBC1 gene including the initiation ATG codon that leads to failure of CYBC1/EROS protein expression and presenting with an unusual clinical manifestation of childhood-onset sarcoidosis-like disease requiring multiple immunosuppressive therapies. We described an abnormal gp91phox protein expression/function in the patient's neutrophils and monocytes (about 50%) and a severely compromised B cell subset (gp91phox < 15%; DHR+ < 4%).Our case-report emphasized the importance of considering a diagnosis of AR-CGD5 deficiency even in absence of typical clinical and laboratory findings.

CRISPR-gene-engineered CYBB knock-out PLB-985 cells, a useful model to study functional impact of X-linked chronic granulomatous disease mutations: application to the G412E X91+-CGD mutation.

Chronic granulomatous disease (CGD) is a rare primary immune disorder caused by mutations in one of the five subunits of the NADPH oxidase complex expressed in phagocytes. Two-thirds of CGD cases are caused by mutations in CYBB that encodes NOX2 or gp91phox. Some rare X91+-CGD point mutations lead to a loss of function but with a normal expression of the mutated NOX2 protein. It is therefore necessary to ensure that this mutation is indeed responsible for the loss of activity in order to make a safe diagnosis for genetic counselling. We previously used the X-CGD PLB-985 cell model of M.C. Dinauer obtained by homologous recombination in the original PLB-985 human myeloid cell line, in order to study the functional impact of such mutations. Although the PLB-985 cell line was originally described by K.A. Tucker et al. in1987 as a distinct cell line isolated from a patient with acute nonlymphocytic leukemia, it is actually identified as a subclone of the HL-60 cells. In order to use a cellular model that meets the quality standard for the functional study of X91+-CGD mutations in CGD diagnosis, we developed our own model using the CRISPR-Cas9 technology in a certified PLB-985 cell line from DSMZ-German Collection of Microorganisms and Cell Cultures. Thanks to this new X-CGD model, we demonstrated that the G412E mutation in NOX2 found in a X91+-CGD patient prohibits access of the electron donor NADPH to its binding site explaining the absence of superoxide production in his neutrophils.

Clinical features and outcomes of patients with chronic granulomatous disease in Taiwan.

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency disease characterized by defective neutrophil killing of microbial pathogens and recurrent infections. We aimed to investigate the clinical, genetic features, treatment, and outcomes in patients with CGD. Pediatric patients diagnosed with CGD from a medical center in Taiwan were enrolled from January 1999 to Oct 2021. Nine pediatric patients with CGD were enrolled: six X-linked (XL) CGD with CYBB gene mutations, three autosomal recessive (AR) CGD with two NCF1 and one CYBA gene mutations.The median age of onset and age of diagnosis was 0.92 and 2.64 years, respectively. Patients with XL-CGD had a younger age of onset (4.6 months vs. 1.83 years, P=0.06) and age of diagnosis (1.71 vs. 8.86 years, P=0.024) than AR-CGD patients. The most common sites of infections were skin and soft tissue abscesses. The most common pathogens were Staphylococcus, Serratia, and Salmonella spp. Prophylactic antibiotics, anti-fungal agents, and interferon-gamma (IFN-γ) were given in 9 (100%), 7 (77.8%), and 8(88.9%) patients, respectively. The mean duration of IFN-γ usage was 5.15 years. One male patient with XL-CGD was successfully treated with hematopoietic stem cell transplantation at 2.2 years. The mortality rate was 11.1%, and the estimated overall survival at 20 years was 66.7%. Staphylococcus aureus, Serratia marcescens, and Salmonella infections are important in Taiwanese CGD patients. Patients with XL-CGD have early disease onset. IFN-γ prophylaxis and prophylactic anti-microbial agents might have an effect on alleviating the infection episodes in CGD patients.

Severe hematopoietic stem cell inflammation compromises chronic granulomatous disease gene therapy

X-linked chronic granulomatous disease (CGD) is associated with defective phagocytosis, life-threatening infections, and inflammatory complications. We performed a clinical trial of lentivirus-based gene therapy in four patients (NCT02757911). Two patients show stable engraftment and clinical benefits, whereas the other two have progressively lost gene-corrected cells. Single-cell transcriptomic analysis reveals a significantly lower frequency of hematopoietic stem cells (HSCs) in CGD patients, especially in the two patients with defective engraftment. These two present a profound change in HSC status, a high interferon score, and elevated myeloid progenitor frequency. We use elastic-net logistic regression to identify a set of 51 interferon genes and transcription factors that predict the failure of HSC engraftment. In one patient, an aberrant HSC state with elevated CEBPβ expression drives HSC exhaustion, as demonstrated by low repopulation in a xenotransplantation model. Targeted treatments to protect HSCs, coupled to targeted gene expression screening, might improve clinical outcomes in CGD.

Imaging Findings in Chronic Granulomatous Disease (CGD).

To describe prevalence of various imaging findings in chronic granulomatous disease (CGD) patients; and find imaging biomarkers for differentiating chest infections caused by different micro-organisms. A retrospective study was conducted on 15 patients (49 scans) with proven CGD. Scans which had a correlative microbiological diagnosis for organisms were included in the analysis. The scans were reviewed by 3 radiologists on a predefined proforma, under the lung parenchymal, airway, pleural, mediastinal, and extrathoracic abnormalities. Analysis of various imaging parameters on a semiquantitative scale was performed, followed by a correlation of each imaging findings with causative organisms. The mean age of presentation was nearly 7 y, with a male preponderance. Definitive proof of causative organisms was obtained in 22 scans. Bacterial infection was found in 7, fungal in 12, tubercular in 2, and viral in 1 scan. Most prevalent thoracic imaging manifestations included lymphadenopathy (commonest), consolidation, nodules, air trapping, and bronchiectasis. Fungal infections showed necrotic conglomerate lymphadenopathy, cavitating nodules, and multilobar consolidation more frequently than bacterial infections (though not statistically significant). Abscesses and lymphadenopathy were the most common extrathoracic manifestations. In patients with CGD, multifocal or multilobar consolidation, mass-like consolidation, cavitating nodules, and conglomerate necrotic lymphadenopathy should alert the radiologist to a possible fungal cause.

Extended Detection in Peripheral Blood Following Infusion of Chronic Granulomatous Disease Patient Autologous Granulocytes Corrected By mRNA Transfection in Patients with Chronic Granulomatous Disease

Patients with Chronic Granulomatous Disease (CGD) have an inherited defect in production of microbicidal oxidants by neutrophils and monocytes that can result in life-threatening infections. Some of these infections, particularly those with mold fungi that invade across tissue boundaries, may persist and progress despite intensive medical therapy. In such a setting there is a long history of published single case and small series reports suggesting that a course of unmatched allogeneic donor granulocyte (gran) transfusions may result in improvement or even resolution of such refractory life-threatening infections in CGD patients. Donors for grans transfusion products are often challenging to find and not HLA-matched. Because of this most CGD patients can be provided only a limited course of grans transfusions before they develop limiting anti-HLA antibodies or lack of available donors. To address this problem, we developed an ex vivo correction of NADPH oxidase defective grans from CGD by mRNA transfection1. Apheresis grans transfected with mRNA restored NADPH oxidase function in vitro and following transplant into immunodeficient mice. Messenger RNA-transfected autologous grans from non-human primates (NHP) were well tolerated and were detected for up to 5 days in NHP peripheral blood following infusion. This pre-clinical work was transitioned to a Phase I, dose-escalating, feasibility and safety study (NIH Protocol 22-I-0001; ClinicalTrials.gov NCT05189925). We report here data from the first X-linked CGD subject infused with autologous gp91 mRNA-transfected grans at the first dose level. The patient received two daily doses of G-CSF before apheresis. From 26 x10e9 total nucleated cells in the apheresis product, ~7x10e9 grans were collected, though only 1.5x10e9 grans were subjected to electroporation transfection with gp91phox mRNA (CELLSCRIPT LLC). A single infusion of 70e6 (10e6 per kg) mRNA-transfected autologous grans where about 57% were DHR+ (40x10e6 DHR positive grans) was well tolerated without any adverse effects. NADPH oxidase functional (DHR+) grans were detected in peripheral blood (PB) at last assessment (72 hrs) after this single infusion of mRNA corrected autologous grans. Serial PB evaluation post infusion from study Subject detected oxidase (DHR)+ grans. Of note, DHR+ grans in Subject PB at ~ 43 hrs after infusion was 0.0037% compared to his pre-infusion DHR assays (0%) (Figure, top row). As a comparator, DHR assays performed pre- and also ~ 40 hrs post infusion of a clinical product of 40x10e9 allogeneic donor grans into another CGD patient being treated clinically for a refractory infection is shown (bottom row). There is 1000 fold more allogeneic DHR+ grans infused for the clinical treatment than the experimental DHR+ mRNA corrected autologous grans infused. Compared to their corresponding absolute neutrophil count, the study Subject (received 40x10e6 DHR+ mRNA transfected autologous grans) had 512 DHR+ neutrophils/ml while the CGD patient (received clinical 40x10e9 allogeneic grans) had 79,560 DHR+ neutrophils/ml. This indicates there are > 6 fold more mRNA-corrected autologous grans in circulation at 43 hrs per cell infused than following the clinical treatment with allogeneic cells infused. Our data demonstrates that mRNA-corrected autologous grans are not only well tolerated but are very well sustained viable and functional in the circulation relative to clinical unmanipulated allogeneic donor grans. Additional patients need to be studied at this dose level and the escalating dose levels to assure that this could be a safe alternative to non-HLA matched allogeneic grans transfusions. Figure shows FACS DHR analysis of peripheral blood from CGD patients after infusion of autologous mRNA-transfected granulocytes 40x106 (top) and donor granulocytes 40x109 (bottom). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Improved Lentiviral In Vivo Gene Therapy for Chronic Granulomatous Disease

Background: Chronic granulomatous disease (CGD) is a congenital immunodeficiency characterized by severe life-threatening infections. CGD patients lack reactive oxygen species (ROS) in phagocytic leukocytes and often develop widespread tissue granulomas. The current treatment for CGD is lifelong antibiotic prophylaxis or hematopoietic stem cell transplantation (HSCT). However, most patients cannot find matched donors, and HSCT is known to have increased risk in CGD patients. Gene therapy has become a promising treatment for CGD. At present, "ex vivo" HSC gene therapy is the widely applied strategy. "In vivo" gene therapy is based on direct delivery of gene therapy vectors into patients, which could overcome the difficulties in HSCT. Methods: We have developed an in vivo CGD gene therapy strategy by intravenous (iv) injection of lentiviral vectors (LVs) carrying an universal EF1a promoter or a myeloid-specific miR223 promoter driving the expression of CYBB (cytochrome B-245 beta chain, or p91-phox) in X-CGD mice (X-CGD; B6.129S-Cybbtm1Din/J). Each mouse received 1x109 transduction units (TU) of LV-CYBB via iv injection after non-myeloablative conditioning. A second injection of 1x109 TU of LV-CYBB after re-conditioning was given 12 weeks later. Results: We developed a chemotherapy conditioning regimen to improve gene delivery efficiency by using busulfan, cyclophosphamide and dexamethasone. The X-CGD mice received two iv injections of LVs, and the expression and function of the LV-CYBB gene were analyzed by intracellular gp91-phox staining and DHR123 assay 4 weeks after each injection. Based on flow cytometry, we detected gp91-phox positive cells in the blood as the following, EF1a: 1.14-5.18% and 5.27-14.36%, and miR223: 1.65-5.17% and 4.59-17.89% after the first and the second injections, respectively, as compared with the WT mice: 79.9-87.6%. Functional analyses of ROS showed the following, EF1a: 1.93-5.2% and 2.39-18.51%, miR223: 2.65-6.67% and 3.75-19.8%, after the first and the second injections respectively, as compared with WT: 72.6-85.31% (Figure A). The LV vector copy number (VCN) in the peripheral blood mononuclear cells was detected by qPCR as the following, EF1a: 1.11-2.32% and 1.35-9.32%, and miR223: 1.01-2.39% and 2.71-8.51% after the first and the second injections, respectively. These results indicated that iv LV injection effectively delivered and expressed the LV-CYBB gene in vivo, and repeated injection of the LVs could increase the gene transfer efficiency in the CGD mice. Importantly, after the second injection, we found no antibody response to the LV particle-associated protein p24. However, after 3-12 months, markedly reduced VCN was found in the blood of the treated mice, suggesting exhaustion of the transgenes in the somatic cells. To improve the gene delivery into HSCs, we treated the mice with AMD3100 to mobilize HSCs into peripheral blood. The mice were treated with chemo-conditioning and AMD3100 prior to LV ivinjection. After four weeks, the gp91-phox positive HSCs of total Sca1+ HSCs in blood with or without (+/-) AMD3100 treatment were examined by flow cytometry, and the results showed the following, EF1a: (-) 9.52-15.43%, (+) 16.1-29.4%, and miR223: (-) 4.49-10.35% and (+) 7.06-16.84%. To confirm stem cell gene transfer, we transplanted the bone marrow cells from the LV-CYBB-treated CGD mice into untreated CGD mice. The VCN in blood was examined before (donor mice) and 4 weeks after (recipient mice) the HSCT, and the results showed the following, EF1a: before 4.49% (in the donor) and after 1.05-2.57% (in the recipients), and miR223: before 4.91% (in the donor) and after 1.04-2.69% (in the recipients) (Figure B). This transplantation study confirmed that the AMD3100 treatment effectively increased iv LV gene delivery into HSCs. Conclusion: We have developed an improved LV iv gene delivery strategy by applying chemo-conditioning and AMD3100 mobilization prior to the injection of the LVs into CGD mice. This in vivo LV gene therapy strategy could potentially be translated into future CGD gene therapy applications. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal