P47phox-deficient Chronic Granulomatous Disease Research Articles

Gene editing of NCF1 loci is associated with homologous recombination and chromosomal rearrangements

CRISPR-based genome editing of pseudogene-associated disorders, such as p47phox-deficient chronic granulomatous disease (p47 CGD), is challenged by chromosomal rearrangements due to presence of multiple targets. We report that interactions between highly homologous sequences that are localized on the same chromosome contribute substantially to post-editing chromosomal rearrangements. We successfully employed editing approaches at the NCF1 gene and its pseudogenes, NCF1B and NCF1C, in a human cell line model of p47 CGD and in patient-derived human hematopoietic stem and progenitor cells. Upon genetic engineering, a droplet digital PCR-based method identified cells with altered copy numbers, spanning megabases from the edited loci. We attributed the high aberration frequency to the interaction between repetitive sequences and their predisposition to recombination events. Our findings emphasize the need for careful evaluation of the target-specific genomic context, such as the presence of homologous regions, whose instability can constitute a risk factor for chromosomal rearrangements upon genome editing.

Targeted knock-in of NCF1 cDNA into the NCF2 locus leads to myeloid phenotypic correction of p47phox-deficient chronic granulomatous disease

The p47phox-deficient chronic granulomatous disease (p47-CGD) is a primary immunodeficiency caused by mutations in the neutrophil cytosolic factor 1 (NCF1) gene, resulting in defective NADPH oxidase function in phagocytes. Due to its complex genomic context, the NCF1 locus is not suited for safe gene editing with current genome editing technologies. Therefore, we developed a targeted NCF1 cDNA knock-in by CRISPR-Cas9 ribonucleoprotein and viral vector template delivery, to restore p47phox expression under the control of the endogenous NCF2 locus. NCF2 encodes for p67phox, an NADPH oxidase subunit that closely interacts with p47phox and is predominantly expressed in myeloid cells. This approach restored p47phox expression and NADPH oxidase function in p47-CGD patient hematopoietic stem and progenitor cells (HSPCs) and in p47phox-deficient mouse HSPCs, with the transgene expression following a myeloid differentiation pattern. Adeno-associated viral vectors performed favorably over integration-deficient lentiviral vectors for template delivery, with fewer off-target integrations and higher correction efficacy in HSPCs. Such myeloid-directed gene editing is promising for clinical CGD gene therapy, as it leads to the co-expression of p47phox and p67phox, ensuring spatiotemporal and near-physiological transgene expression in myeloid cells.

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.

Preclinical Optimization and Safety Studies of a New Lentiviral Gene Therapy for p47phox-Deficient Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is an inherited blood disorder of phagocytic cells that renders patients susceptible to infections and inflammation. A recent clinical trial of lentiviral gene therapy for the most frequent form of CGD, X-linked, has demonstrated stable correction over time, with no adverse events related to the gene therapy procedure. We have recently developed a parallel lentiviral vector for p47phox-deficient CGD (p47phoxCGD), the second most common form of this disease. Using this vector, we have observed biochemical correction of CGD in a mouse model of the disease. In preparation for clinical trial approval, we have performed standardized preclinical studies following Good Laboratory Practice (GLP) principles, to assess the safety of the gene therapy procedure. We report no evidence of adverse events, including mutagenesis and tumorigenesis, in human hematopoietic stem cells transduced with the lentiviral vector. Biodistribution studies of transduced human CD34+ cells indicate that the homing properties or engraftment ability of the stem cells is not negatively affected. CD34+ cells derived from a p47phoxCGD patient were subjected to an optimized transduction protocol and transplanted into immunocompromised mice. After the procedure, patient-derived neutrophils resumed their function, suggesting that gene correction was successful. These studies pave the way to a first-in-man clinical trial of lentiviral gene therapy for the treatment of p47phoxCGD.

Lentiviral gene therapy rescues p47phox chronic granulomatous disease and the ability to fight Salmonella infection in mice

Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency disorder characterised by recurrent and often life-threatening infections and hyperinflammation. It is caused by defects of the phagocytic NADPH oxidase, a multicomponent enzyme system responsible for effective pathogen killing. A phase I/II clinical trial of lentiviral gene therapy is underway for the most common form of CGD, X-linked, caused by mutations in the gp91phox subunit of the NADPH oxidase. We propose to use a similar strategy to tackle p47phox-deficient CGD, caused by mutations in NCF1, which encodes the p47phox cytosolic component of the enzymatic complex. We generated a pCCLCHIM-p47phox lentiviral vector, containing the chimeric Cathepsin G/FES myeloid promoter and a codon-optimised version of the human NCF1 cDNA. Here we show that transduction with the pCCLCHIM-p47phox vector efficiently restores p47phox expression and biochemical NADPH oxidase function in p47phox-deficient human and murine cells. We also tested the ability of our gene therapy approach to control infection by challenging p47phox-null mice with Salmonella Typhimurium, a leading cause of sepsis in CGD patients, and found that mice reconstituted with lentivirus-transduced hematopoietic stem cells had a reduced bacterial load compared with untreated mice. Overall, our results potentially support the clinical development of a gene therapy approach using the pCCLCHIM-p47phox vector.

CRISPR-Directed Therapeutic Correction at the NCF1 Locus Is Challenged by Frequent Incidence of Chromosomal Deletions

Resurrection of non-processed pseudogenes may increase the efficacy of therapeutic gene editing, upon simultaneous targeting of a mutated gene and its highly homologous pseudogenes. To investigate the potency of this approach for clinical gene therapy of human diseases, we corrected a pseudogene-associated disorder, the immunodeficiency p47phox-deficient chronic granulomatous disease (p47phox CGD), using clustered regularly interspaced short palindromic repeats-associated nuclease Cas9 (CRISPR-Cas9) to target mutated neutrophil cytosolic factor 1 (NCF1). Being separated by less than two million base pairs, NCF1 and two pseudogenes are closely co-localized on chromosome 7. In healthy people, a two-nucleotide GT deletion (ΔGT) is present in the NCF1B and NCF1C pseudogenes only. In the majority of patients with p47phox CGD, the NCF1 gene is inactivated due to a ΔGT transfer from one of the two non-processed pseudogenes. Here we demonstrate that concurrent targeting and correction of mutated NCF1 and its pseudogenes results in therapeutic CGD phenotype correction, but also causes potentially harmful chromosomal deletions between the targeted loci in a p47phox-deficient CGD cell line model. Therefore, development of genome-editing-based treatment of pseudogene-related disorders mandates thorough safety examination, as well as technological advances, limiting concurrent induction of multiple double-strand breaks on a single chromosome.

INFECTION OR INFLAMMATION? RAPIDLY PROGRESSIVE PULMONARY CONSOLIDATION IN A PATIENT WITH CHRONIC GRANULOMATOUS DISEASE

SESSION TITLE: Monday Medical Student/Resident Case Report Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: 10/21/2019 02:30 PM - 03:15 PM INTRODUCTION: Fever in patients with chronic granulomatous disease (CGD) poses a unique diagnostic challenge for clinicians. We present a case of progressive right middle lobe consolidation in a patient with CGD which was determined to be secondary to Burkholderia multivorans after extensive investigation. CASE PRESENTATION: A 24 year old woman with p47phox-deficient CGD, compliant with interferon and antimicrobial prophylactic therapy, was admitted for the third time in one month with fevers, dyspnea, productive cough, and pleuritic chest pain. At her first admission, imaging showed right hilar lymphadenopathy with multiple pulmonary nodules. Bronchoscopy with lavage and endobronchial ultrasound was notable for Actinomyces, and high dose amoxicillin was prescribed. She was re-admitted for persistent symptoms, with repeat imaging showing new mediastinal consolidation in the right middle lobe. Bronchoscopy with trans-bronchial biopsies were unrevealing. She was discharged on cefepime and voriconazole empirically. She was readmitted due to persistent symptoms and imaging showed worsening right middle lobe consolidation and new pulmonary nodules, and vancomycin was added. Pathology obtained from bronchoscopy with cryobiopsy showed focal interstitial non-caseating granulomas, and oral steroids were initiated for possible CGD flare. Given persistent symptoms refractory to medical therapy, right middle lobe resection was performed. Pathology showed extensive multifocal caseating granulomas, and tissue cultures showed Burkholderia multivorans, sensitive to ciprofloxacin. Follow-up CT scan one month later showed improvement in pulmonary nodules and no further consolidation. DISCUSSION: CGD is an immunodeficiency syndrome characterized by inheritable defects of NADPH oxidase, leading to the inability of phagocytic cells to destroy ingested organisms. Patients are often diagnosed in childhood due to recurrent life-threatening infections or complications from granuloma formation. Pulmonary involvement is common, and infectious etiologies such as Aspergillus are responsible for 40-85% of all lung disease in CGD patients. Noninfectious pulmonary complications include granuloma formation, which may be triggered by incompletely resolved or recurrent infection, making the distinction of sterile versus infected granulomas harder. Isolation of offending organisms is crucial, but only occurs in about 52% of cases (1). While the use of antimicrobial prophylaxis has improved mortality of CGD patients, respiratory involvement is a poor prognostic factor, rendering early identification of etiology critical. CONCLUSIONS: In patients with chronic granulomatous disease with persistent pulmonary symptoms refractory to therapy, aggressive tissue sampling including surgical biopsy should be pursued to identify etiology. Reference #1: Mahdaviani, Seyed Alireza, et al. "Pulmonary manifestations of chronic granulomatous disease.” Expert Review of Clinical Immunology 9.2 (2013): 153-160. Reference #2: Salvator, Hélène, et al. "Pulmonary manifestations in adult patients with chronic granulomatous disease.” European Respiratory Journal 45.6 (2015): 1613-1623. DISCLOSURES: No relevant relationships by Michael Agatstein, source=Web Response No relevant relationships by Deepika Kulkarni, source=Web Response No relevant relationships by Seetha Lakshmi, source=Web Response

Novel Diagnostic Tool for p47phox-Deficient Chronic Granulomatous Disease Patient and Carrier Detection

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations of the phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Autosomal recessive p47phox-deficient CGD (p47phox CGD) is the second most frequent form of the disease in western countries, and more than 94% of patients have a disease-causing dinucleotide deletion (ΔGT) in the neutrophil cytosolic factor 1 (NCF1) gene. The ΔGT mutation is most likely transferred onto the NCF1 from one of its two pseudogenes co-localized on the same chromosome. The presence of NCF1 pseudogenes in healthy individuals makes the genetic diagnostics of ΔGT p47phox CGD challenging, as it requires the distinction between ΔGT in NCF1 and in the two pseudogenes. We have developed a diagnostic tool for the identification of p47phox CGD based on PCR co-amplification of NCF1 and its pseudogenes, followed by band intensity quantification of restriction fragment length polymorphism products. The single-day, reliable p47phox CGD diagnostics allow for robust discrimination of homozygous ΔGT p47phox CGD patients from heterozygous carriers and healthy individuals, as well as for monitoring gene therapy efficacy.

CRISPR/Cas9-generated p47phox-deficient cell line for Chronic Granulomatous Disease gene therapy vector development

Development of gene therapy vectors requires cellular models reflecting the genetic background of a disease thus allowing for robust preclinical vector testing. For human p47phox-deficient chronic granulomatous disease (CGD) vector testing we generated a cellular model using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to introduce a GT-dinucleotide deletion (ΔGT) mutation in p47phox encoding NCF1 gene in the human acute myeloid leukemia PLB-985 cell line. CGD is a group of hereditary immunodeficiencies characterized by impaired respiratory burst activity in phagocytes due to a defective phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In Western countries autosomal-recessive p47phox-subunit deficiency represents the second largest CGD patient cohort with unique genetics, as the vast majority of p47phox CGD patients carries ΔGT deletion in exon two of the NCF1 gene. The established PLB-985 NCF1 ΔGT cell line reflects the most frequent form of p47phox-deficient CGD genetically and functionally. It can be differentiated to granulocytes efficiently, what creates an attractive alternative to currently used iPSC models for rapid testing of novel gene therapy approaches.

Gene-edited pseudogene resurrection corrects p47phox-deficient chronic granulomatous disease

AbstractPseudogenes are duplicated genes with mutations rendering them nonfunctional. For single-gene disorders with homologous pseudogenes, the pseudogene might be a target for genetic correction. Autosomal-recessive p47phox-deficient chronic granulomatous disease (p47-CGD) is a life-threatening immune deficiency caused by mutations in NCF1, a gene with 2 pseudogenes, NCF1B and NCF1C. The most common NCF1 mutation, a GT deletion (ΔGT) at the start of exon 2 (>90% of alleles), is constitutive to NCF1B and NCF1C. NCF1 ΔGT results in premature termination, undetectable protein expression, and defective production of antimicrobial superoxide in neutrophils. We examined strategies for p47-CGD gene correction using engineered zinc-finger nucleases targeting the exon 2 ΔGT in induced pluripotent stem cells or CD34+ hematopoietic stem cells derived from p47-CGD patients. Correction of ΔGT in NCF1 pseudogenes restores oxidase function in p47-CGD, providing the first demonstration that targeted restoration of pseudogene function can correct a monogenic disorder.

Different unequal cross-over events between NCF1 and its pseudogenes in autosomal p47phox-deficient chronic granulomatous disease

Chronic granulomatous disease (CGD) is a rare congenital disorder in which phagocytes cannot generate superoxide (O2−) and other microbicidal oxidants due to mutations in one of the five components of the O2−-generating NADPH oxidase complex. The most common autosomal subtype of CGD is caused by mutations in NCF1, encoding the NADPH subunit p47phox. Usually, these mutations are the result of unequal exchange of chromatid between NCF1 and one of its two pseudogenes. We have now investigated in detail the breakpoints within or between these (pseudo) NCF1 genes in 43 families with p47phox-deficient CGD by means of multiplex ligase-dependent probe amplification (MLPA). In 24 families the patients totally lacked NCF1 sequences, indicating that in these families the cross-over points are located between NCF1 and its pseudogenes. Six other families were compound heterozygous for a total NCF1 deletion and another mutation in NCF1 on the other allele. In 8 families, the patients lacked NCF1 exons 1–4 but had retained NCF1 exons 6–10, indicating that a cross-over point is located within NCF1 between exons 4 and 6. Similarly, in 4 families a cross-over point was located within NCF1 between exons 2 and 4. Similar cross-overs, in heterozygous form, were observed in family members of the patients. Several patients were compound heterozygous for total and partial NCF1 deletions. Thus, at least three different cross-over points exist within the NCF1 gene cluster, indicating that autosomal p47phox-deficient CGD is genetically heterogeneous but can be dissected in detail by MLPA.

Signed outside: a surface marker system for transgenic cytoplasmic proteins

Chronic granulomatous disease is a primary immunodeficiency, comprising five molecular defects, characterized by an impaired respiratory burst activity of myeloid cells. We are currently developing a gene therapy vector for the p47phox-deficient form of chronic granulomatous disease. Classic intracellular immunostaining of the cytoplasmic p47phox transgene product, however, interferes with respiratory burst activity. In this study we report a new system for measuring p47phox expression: A single open reading frame encoding the surface marker protein ΔLNGFR (truncated low-affinity nerve growth factor receptor) linked to the p47phox transgene by the 2A oligopeptide coexpression technology. Translation generates two discrete products: p47phox localizing to the cytoplasm and 'ΔLNGFR-2A' localizing to the cell surface. Six weeks after transplantation of transduced autologous hematopoietic stem cells into p47-/- mice, the intracellular p47phox fluorescence-activated cell sorting (FACS) signal intensities corresponded to surface ΔLNGFR staining in monocytes, B cells, T cells and Sca I+ bone marrow cells in vivo. The p47phox cleavage product restored nicotinamide adenine dinucleotide phosphate-oxidase activity in granulocytes differentiated from transduced p47phox-/- murine hematopoietic stem cells ex vivo, in murine granulocytes/monocytes in vivo, and in transduced human monocyte derived macrophages from p47phox-deficient chronic granulomatous disease patients. In conclusion, this new marker system allows highly efficient, indirect detection of cytoplasmic transgene products by FACS surface staining.

A novel mutation in NCF1 in an adult CGD patient with a liver abscess as first presentation

Chronic granulomatous disease (CGD) is an immunodeficiency caused by defects in the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase complex and is usually diagnosed in early childhood. CGD patients suffer from severe, recurrent infections with bacteria, fungi and yeasts. We report a 25-year-old female with protracted fever because of a Staphylococcus aureus liver abscess, which did not resolve until breakthrough into the stomach. Despite her age, CGD was considered on diagnosis on the basis of the clinical symptoms. Analysis of the NADPH-oxidase activity confirmed CGD as the underlying condition. Western blotting revealed the absence of p47(phox) and subsequent sequencing of the p47(phox)-encoding gene, neutrophil cytosolic factor (NCF1), identified a deletion of 837C in the maternal NCF1 allele. The paternal allele contained a stopcodon because of a conversion between NCF1 and one of its PsiNCF1 pseudogenes. The patient had one novel mutation, c.837delC, and one conversion in NCF1, resulting in the complete absence of the p47(phox) component of the NADPH-oxidase complex. This p47(phox)-deficient CGD patient had the highest age at diagnosis reported thus far.

Drug-selected complete restoration of superoxide generation in Epstein-Barr virus-transformed B cells from p47phox-deficient chronic granulomatous disease patients by using a bicistronic retrovirus vector encoding a human multi-drug resistance gene (MDR1) and the p47phox gene.

Chronic granulomatous disease (CGD) is a group of disorders characterized by the failure of phagocytes to produce superoxide. One-third of the cases of CGD in the USA and Europe results from defects in the gene encoding p47phox, a cytoplasmic component of NADPH oxidase for superoxide generation. In this study, we constructed the bicistronic retrovirus vector Ha-MDR-IRES-p47, which carries cDNAs for a human multi-drug-resistance gene (MDR1) and p47phox. The amphotropic retroviral producer cells were generated, and the supernatant of the producer cells was used to transduce Epstein-Barr virus-transformed B (EBV-B) cells, established from B cells of p47phox-deficient CGD patients, as an in vitro model of gene therapy for p47phox-deficient CGD. The transduced cells expressed both P-glycoprotein and p47phox protein, and the expression levels were increased after appropriate vincristine selection. The levels of superoxide production in the vincristine-selected cells were increased to a level similar to normal EBV-B cells. This result suggests that it is possible to achieve 100% correction of the CGD defect in p47phox-deficient EBV-B cells by using the bicistronic vector. This strategy could be employed not only in vitro, but also in vivo, in the gene therapy of a number of inherited diseases.

Peripheral blood progenitors as a target for genetic correction of p47phox-deficient chronic granulomatous disease.

Peripheral blood contains hematopoietic progenitors (PBHPs), which can be harvested in clinically relevant amounts by apheresis. PBHPs have been used as a source of progenitors alternative to marrow for autologous transplantation following intensive chemotherapy. We have determined culture conditions for growth and differentiation of PBHPs to the mature myeloid phenotype, which in the present study are employed to demonstrate the functional correction of an inherited disorder of myeloid cells in retrovirus-transduced human primary hematopoietic progenitors. Patients with chronic granulomatous disease (CGD) suffer from recurrent life-threatening infections because blood phagocytes fail to produce microbicidal superoxide (O2-.). One-third of the cases of CGD result from defects in the gene encoding p47phox, a cytoplasmic oxidase component required for O2-. generation. In the present study, a replication-defective retrovirus encoding p47phox was used to transduce PBHPs from patients with p47phox-deficient CGD, which resulted in significant correction of O2-. generation when PBHPs were differentiated to mature neutrophils and monocytes. This study provides a model for use of PBHPs in development of gene therapy for diseases affecting bone marrow.

O2- production by B lymphocytes lacking the respiratory burst oxidase subunit p47phox after transfection with an expression vector containing a p47phox cDNA.

The respiratory burst oxidase of phagocytes and B lymphocytes is a complicated enzyme that catalyzes the one-electron reduction of oxygen by NADPH. It is responsible for the O2- production that occurs when these cells are exposed to phorbol 12-myristate 13-acetate or other appropriate stimuli. The activity of this enzyme is greatly decreased or absent in patients with chronic granulomatous disease, an inherited disorder characterized by a severe defect in host defense against bacteria and fungi. In every chronic granulomatous disease patient studied to date, an abnormality has been found in a gene encoding one of four components of the respiratory burst oxidase: the membrane protein p22phox or gp91phox, or the cytosolic protein p47phox or p67phox. We report here that O2- production was partly restored to phorbol 12-myristate 13-acetate-stimulated Epstein-Barr virus-transformed B lymphocytes from a patient with p47phox-deficient chronic granulomatous disease by transfection with an expression plasmid containing a p47phox cDNA inserted in the sense direction. No detectable O2- was produced by untransfected p47phox-deficient lymphocytes or by p47phox-deficient lymphocytes transfected with an antisense plasmid. The finding that O2- can be produced by p47phox-deficient B lymphocytes after the transfer of a p47phox cDNA into the deficient cells suggests that this system could be useful for studying the function of mutant p47phox proteins in whole cells.

Retroviral expression of recombinant p47phox protein by Epstein-Barr virus-transformed B lymphocytes from a patient with autosomal chronic granulomatous disease

Patients with chronic granulomatous disease (CGD) have recurrent infections resulting from a failure of phagocytic cells to produce superoxide. One third of CGD patients have an autosomal gene defect resulting in absence of p47phox protein, a cytoplasmic component critical to superoxide production by phagocytic cells. cDNA encoding p47phox has been cloned and recombinant p47phox (rp47phox) restores superoxide-generating activity to a cell-free assay containing cell membranes and cytosol from p47phox-deficient CGD neutrophils. The goal of the present study was to determine the feasibility of retrovirus mediated expression of rp47phox in the HL60 and U937 human hematopoietic cell lines, and in an Epstein-Barr virus transformed B- lymphocyte cell line (EBV-BCL) derived from a p47phox-deficient CGD patient. Normal EBV-BCL contain p47phox and generate small amounts of superoxide, while this CGD EBV-BCL lacks any detectable p47phox protein. Defective amphotropic retrovirus containing p47phox sequence inserted in the LXSN vector in sense and antisense orientations were used to transduce HL60, U937, and CGD EBV-BCL. p47phox mRNA sequence was detected in cells transduced with either sense or antisense retroviral constructs while rp47phox protein was detected only with the sense construct. The amount of rp47phox protein produced within these cells was greater than the native p47phox present in uninduced HL60 or U937 cells, but substantially less than that present in normal neutrophils, induced HL60 cells, or even normal EBV-BCL. Differentiation of transduced HL60 cells and the associated production of native p47phox in response to dimethyl sulfoxide was not affected. These studies demonstrate that retrovirus constructs can be used to mediate stable expression of rp47phox protein in human hematopoietic cell lines and can restore rp47phox protein within the cytosol of p47phox-deficient EBV-BCL from patients with CGD.

Retroviral expression of recombinant p47phox protein by Epstein-Barr virus-transformed B lymphocytes from a patient with autosomal chronic granulomatous disease

Patients with chronic granulomatous disease (CGD) have recurrent infections resulting from a failure of phagocytic cells to produce superoxide. One third of CGD patients have an autosomal gene defect resulting in absence of p47phox protein, a cytoplasmic component critical to superoxide production by phagocytic cells. cDNA encoding p47phox has been cloned and recombinant p47phox (rp47phox) restores superoxide-generating activity to a cell-free assay containing cell membranes and cytosol from p47phox-deficient CGD neutrophils. The goal of the present study was to determine the feasibility of retrovirus mediated expression of rp47phox in the HL60 and U937 human hematopoietic cell lines, and in an Epstein-Barr virus transformed B-lymphocyte cell line (EBV-BCL) derived from a p47phox-deficient CGD patient. Normal EBV-BCL contain p47phox and generate small amounts of superoxide, while this CGD EBV-BCL lacks any detectable p47phox protein. Defective amphotropic retrovirus containing p47phox sequence inserted in the LXSN vector in sense and antisense orientations were used to transduce HL60, U937, and CGD EBV-BCL. p47phox mRNA sequence was detected in cells transduced with either sense or antisense retroviral constructs while rp47phox protein was detected only with the sense construct. The amount of rp47phox protein produced within these cells was greater than the native p47phox present in uninduced HL60 or U937 cells, but substantially less than that present in normal neutrophils, induced HL60 cells, or even normal EBV-BCL. Differentiation of transduced HL60 cells and the associated production of native p47phox in response to dimethyl sulfoxide was not affected. These studies demonstrate that retrovirus constructs can be used to mediate stable expression of rp47phox protein in human hematopoietic cell lines and can restore rp47phox protein within the cytosol of p47phox-deficient EBV-BCL from patients with CGD.