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1.
Front Cell Dev Biol ; 8: 608600, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33365312

RESUMEN

Neutrophils are the first cells recruited at the site of infections, where they phagocytose the pathogens. Inside the phagosome, pathogens are killed by proteolytic enzymes that are delivered to the phagosome following granule fusion, and by reactive oxygen species (ROS) produced by the NADPH oxidase. The NADPH oxidase complex comprises membrane proteins (NOX2 and p22phox), cytoplasmic subunits (p67phox, p47phox, and p40phox) and the small GTPase Rac. These subunits assemble at the phagosomal membrane upon phagocytosis. In resting neutrophils the catalytic subunit NOX2 is mainly present at the plasma membrane and in the specific granules. We show here that NOX2 is also present in early and recycling endosomes in human neutrophils and in the neutrophil-like cell line PLB-985 expressing GFP-NOX2. In the latter cells, an increase in NOX2 at the phagosomal membrane was detected by live-imaging after phagosome closure, probably due to fusion of endosomes with the phagosome. Using super-resolution microscopy in PLB-985 WT cells, we observed that NOX2 forms discrete clusters in the plasma membrane. The number of clusters increased during frustrated phagocytosis. In PLB-985NCF1ΔGT cells that lack p47phox and do not assemble a functional NADPH oxidase, the number of clusters remained stable during phagocytosis. Our data suggest a role for p47phox and possibly ROS production in NOX2 recruitment at the phagosome.

2.
Mol Ther Methods Clin Dev ; 17: 936-943, 2020 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-32420407

RESUMEN

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 p47 phox -deficient chronic granulomatous disease (p47 phox 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 p47 phox 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 p47 phox -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.

3.
Front Immunol ; 10: 2236, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31681257

RESUMEN

Background: Chronic granulomatous disease (CGD) is caused by a malfunctioning nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex in phagocytes, leading to impaired bacterial and fungal killing and hyperinflammation. Objective: To characterize macrophage subsets and cytokine/chemokine signaling loops involved in CGD tissue hyperinflammation. Methods: Cytokine/chemokine production and surface marker expression were analyzed in inflamed tissue of four CGD patients and compared to cytokine/chemokine released by CGD macrophages upon priming to different macrophage subpopulations. Furthermore, the re-priming capacity of CGD pro-inflammatory M1 to M2a anti-inflammatory macrophages was evaluated. Results: In human CGD inflammatory tissue, IL-18 and IFN-γ were detected in significant quantity. Immunofluorescence analysis identified macrophages as one source of IL-18 in inflamed tissue. In vitro, CGD macrophages could be primed and re-primed into all inflammatory/anti-inflammatory macrophage subpopulations. IL-18 was also released by M1 CGD and control macrophages. Conclusion: CGD pro-inflammatory M1 macrophages remain M1 primed in vivo. As CGD M1 macrophages can be re-primed to anti-inflammatory M2a phenotype in vitro, macrophages are kept in M1 state in vivo by a persistent pro-inflammatory environment. Our results suggest a paracrine signaling loop between M1 macrophage derived IL-18 and non-macrophage derived IFN-γ maintaining macrophage pro-inflammatory activity in CGD tissue.


Asunto(s)
Enfermedad Granulomatosa Crónica/inmunología , Interferón gamma/inmunología , Interleucina-18/inmunología , Macrófagos/inmunología , Comunicación Paracrina/inmunología , Transducción de Señal/inmunología , Adolescente , Adulto , Niño , Femenino , Enfermedad Granulomatosa Crónica/genética , Enfermedad Granulomatosa Crónica/patología , Humanos , Lactante , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Interferón gamma/genética , Interleucina-18/genética , Macrófagos/patología , Masculino , Comunicación Paracrina/genética , Transducción de Señal/genética
4.
J Clin Immunol ; 39(3): 298-308, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30838481

RESUMEN

DOCK2 is a guanine-nucleotide-exchange factor for Rac proteins. Activated Rac serves various cellular functions including the reorganization of the actin cytoskeleton in lymphocytes and neutrophils and production of reactive oxygen species in neutrophils. Since 2015, six unrelated patients with combined immunodeficiency and early-onset severe viral infections caused by bi-allelic loss-of-function mutations in DOCK2 have been described. Until now, the function of phagocytes, specifically neutrophils, has not been assessed in human DOCK2 deficiency. Here, we describe a new kindred with four affected siblings harboring a homozygous splice-site mutation (c.2704-2 A > C) in DOCK2. The mutation results in alternative splicing and a complete loss of DOCK2 protein expression. The patients presented with leaky severe combined immunodeficiency or Omenn syndrome. The novel mutation affects EBV-B cell migration and results in NK cell dysfunction similar to previous observations. Moreover, both cytoskeletal rearrangement and reactive oxygen species production are partially impaired in DOCK2-deficient neutrophils.


Asunto(s)
Linfocitos B/inmunología , Proteínas Activadoras de GTPasa/genética , Factores de Intercambio de Guanina Nucleótido/genética , Células Asesinas Naturales/inmunología , Neutrófilos/inmunología , Eliminación de Secuencia/genética , Inmunodeficiencia Combinada Grave/genética , Empalme Alternativo/genética , Humanos , Estrés Oxidativo , Linaje
5.
Mol Ther Methods Clin Dev ; 13: 274-278, 2019 Jun 14.
Artículo en Inglés | MEDLINE | ID: mdl-30859112

RESUMEN

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations of the phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Autosomal recessive p47 phox -deficient CGD (p47 phox 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 p47 phox 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 p47 phox 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 p47 phox 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.

6.
Matters (Zur) ; 20182018.
Artículo en Inglés | MEDLINE | ID: mdl-31008103

RESUMEN

A recent gamma-retroviral clinical Chronic Granulomatous Disease (CGD) gene therapy (GT) trial achieved proof-of-concept but was accompanied by activation of oncogenes and transgene silencing. The ubiquitous chromatin opening element (UCOE) comprises the sequences of two divergently oriented house-keeping gene promoters and is known to have anti-silencing properties. In a screen we identified two novel UCOE constructs that prevent adjacent promoter methylation in P19 cells. Experiments were continued with the shorter UCOE constructs in induced pluripotent stem cells (iPSC) derived from a p47phox-deficient CGD patient. The iPSC line was transduced with the lentiviral GT vectors expressing P47 under the constitutively active SFFV promoter with UCOE element (UCOE_SF) and without UCOE element (SF) adjacent to the SFFV promoter. The iPSC were expanded before propagation towards neutrophils. 20 days after transduction the UCOE_SF vector was protected from methylation in iPSC as previously shown in P19 cells, whereas the SF vector was heavily methylated in iPSC. The UCOE_SF vector maintained stable transgene expression in iPSC, macrophages and neutrophils, whereas the SF vector was strongly silenced. The UCOE_SF vector stably restored ROS production in neutrophils, whereas for the SF vector the count of ROS producing cells was marginal. To conclude, we have shown that the prevention of transgene silencing by UCOE is functionally and mechanistically preserved upon terminal neutrophil differentiation.

7.
Sci Rep ; 7: 44187, 2017 03 13.
Artículo en Inglés | MEDLINE | ID: mdl-28287132

RESUMEN

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.


Asunto(s)
Sistemas CRISPR-Cas , Terapia Genética/métodos , Vectores Genéticos , Enfermedad Granulomatosa Crónica , Secuencia de Bases , Enfermedad Granulomatosa Crónica/genética , Enfermedad Granulomatosa Crónica/metabolismo , Enfermedad Granulomatosa Crónica/patología , Enfermedad Granulomatosa Crónica/terapia , Células HL-60 , Humanos , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Eliminación de Secuencia
8.
J Allergy Clin Immunol ; 139(1): 212-219.e3, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-27458052

RESUMEN

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymatic disorder of red blood cells in human subjects, causing hemolytic anemia linked to impaired nicotinamide adenine dinucleotide phosphate (NADPH) production and imbalanced redox homeostasis in erythrocytes. Because G6PD is expressed by a variety of hematologic and nonhematologic cells, a broader clinical phenotype could be postulated in G6PD-deficient patients. We describe 3 brothers with severe G6PD deficiency and susceptibility to bacterial infection. OBJECTIVE: We sought to study the molecular pathophysiology leading to susceptibility to infection in 3 siblings with severe G6PD deficiency. METHODS: Blood samples of 3 patients with severe G6PD deficiency were analyzed for G6PD enzyme activity, cellular oxidized nicotinamide adenine dinucleotide phosphate/NADPH levels, phagocytic reactive oxygen species production, neutrophil extracellular trap (NET) formation, and neutrophil elastase translocation. RESULTS: In these 3 brothers strongly reduced NADPH oxidase function was found in granulocytes, leading to impaired NET formation. Defective NET formation has thus far been only observed in patients with the NADPH oxidase deficiency chronic granulomatous disease, who require antibiotic and antimycotic prophylaxis to prevent life-threatening bacterial and fungal infections. CONCLUSION: Because severe G6PD deficiency can be a phenocopy of chronic granulomatous disease with regard to the cellular and clinical phenotype, careful evaluation of neutrophil function seems mandatory in these patients to decide on appropriate anti-infective preventive measures. Determining the level of G6PD enzyme activity should be followed by analysis of reactive oxygen species production and NET formation to decide on required antibiotic and antimycotic prophylaxis.


Asunto(s)
Susceptibilidad a Enfermedades , Trampas Extracelulares/metabolismo , Deficiencia de Glucosafosfato Deshidrogenasa , Infecciones Bacterianas , Niño , Eritrocitos/metabolismo , Glucosafosfato Deshidrogenasa/genética , Glucosafosfato Deshidrogenasa/metabolismo , Deficiencia de Glucosafosfato Deshidrogenasa/diagnóstico , Deficiencia de Glucosafosfato Deshidrogenasa/genética , Deficiencia de Glucosafosfato Deshidrogenasa/metabolismo , Granulocitos/metabolismo , Humanos , Lactante , Elastasa de Leucocito/metabolismo , Masculino , NADP/metabolismo , Especies Reactivas de Oxígeno/metabolismo
9.
J Cereb Blood Flow Metab ; 37(8): 2833-2847, 2017 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-27789786

RESUMEN

Near-infrared fluorescence (NIRF) imaging enables non-invasive monitoring of molecular and cellular processes in live animals. Here we demonstrate the suitability of NIRF imaging to investigate the neutrophil response in the brain after transient middle cerebral artery occlusion (tMCAO). We established procedures for ex vivo fluorescent labelling of neutrophils without affecting their activation status. Adoptive transfer of labelled neutrophils in C57BL/6 mice before surgery resulted in higher fluorescence intensities over the ischaemic hemisphere in tMCAO mice with NIRF imaging when compared with controls, corroborated by ex vivo detection of labelled neutrophils using fluorescence microscopy. NIRF imaging showed that neutrophils started to accumulate immediately after tMCAO, peaking at 18 h, and were still visible until 48 h after reperfusion. Our data revealed accumulation of neutrophils also in extracranial tissue, indicating damage in the external carotid artery territory in the tMCAO model. Antibody-mediated inhibition of α4-integrins did reduce fluorescence signals at 18 and 24, but not at 48 h after reperfusion, compared with control treatment animals. Antibody treatment reduced cerebral lesion volumes by 19%. In conclusion, the non-invasive nature of NIRF imaging allows studying the dynamics of neutrophil recruitment and its modulation by targeted interventions in the mouse brain after transient experimental cerebral ischaemia.


Asunto(s)
Encéfalo/diagnóstico por imagen , Arteria Carótida Externa/diagnóstico por imagen , Ataque Isquémico Transitorio/diagnóstico por imagen , Monitoreo Fisiológico/métodos , Infiltración Neutrófila , Espectroscopía Infrarroja Corta/métodos , Animales , Encéfalo/irrigación sanguínea , Encéfalo/inmunología , Arteria Carótida Externa/inmunología , Modelos Animales de Enfermedad , Ataque Isquémico Transitorio/inmunología , Masculino , Ratones Endogámicos C57BL , Microscopía Fluorescente
10.
Pediatr Allergy Immunol ; 28(2): 124-134, 2017 03.
Artículo en Inglés | MEDLINE | ID: mdl-27612320

RESUMEN

Phagocytic neutrophil granulocytes are among the first immune cells active at sites of infection, forming an important first-line defense against invading microorganisms. Congenital immune defects concerning these phagocytes may be due to reduced neutrophil numbers or function. Management of affected patients depends on the type and severity of disease. Here, we provide an overview of causes and treatment of diseases associated with congenital neutropenia, as well as defects of the phagocytic respiratory burst.


Asunto(s)
Enfermedad Granulomatosa Crónica/inmunología , Síndromes de Inmunodeficiencia/inmunología , Neutropenia/congénito , Neutrófilos/inmunología , Fagocitosis , Verrugas/inmunología , Animales , Profilaxis Antibiótica , Diferenciación Celular , Síndromes Congénitos de Insuficiencia de la Médula Ósea , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Enfermedad Granulomatosa Crónica/terapia , Trasplante de Células Madre Hematopoyéticas , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunidad Innata , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/terapia , Mielopoyesis/genética , Neutropenia/genética , Neutropenia/inmunología , Neutropenia/terapia , Fagocitosis/genética , Enfermedades de Inmunodeficiencia Primaria , Estallido Respiratorio/genética , Verrugas/genética , Verrugas/terapia
11.
J Allergy Clin Immunol ; 138(1): 219-228.e9, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-26853280

RESUMEN

BACKGROUND: Defects in phagocytic nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) function cause chronic granulomatous disease (CGD), a primary immunodeficiency characterized by dysfunctional microbicidal activity and chronic inflammation. OBJECTIVE: We sought to study the effect of chronic inflammation on the hematopoietic compartment in patients and mice with X-linked chronic granulomatous disease (X-CGD). METHODS: We used immunostaining and functional analyses to study the hematopoietic compartment in patients with CGD. RESULTS: An analysis of bone marrow cells from patients and mice with X-CGD revealed a dysregulated hematopoiesis characterized by increased numbers of hematopoietic progenitor cells (HPCs) at the expense of repopulating hematopoietic stem cells (HSCs). In patients with X-CGD, there was a clear reduction in the proportion of HSCs in bone marrow and peripheral blood, and they were also more rapidly exhausted after in vitro culture. In mice with X-CGD, increased cycling of HSCs, expansion of HPCs, and impaired long-term engraftment capacity were found to be associated with high concentrations of proinflammatory cytokines, including IL-1ß. Treatment of wild-type mice with IL-1ß induced enhanced cell-cycle entry of HSCs, expansion of HPCs, and defects in long-term engraftment, mimicking the effects observed in mice with X-CGD. Inhibition of cytokine signaling in mice with X-CGD reduced HPC numbers but had only minor effects on the repopulating ability of HSCs. CONCLUSIONS: Persistent chronic inflammation in patients with CGD is associated with hematopoietic proliferative stress, leading to a decrease in the functional activity of HSCs. Our observations have clinical implications for the development of successful autologous cell therapy approaches.


Asunto(s)
Enfermedad Granulomatosa Crónica/metabolismo , Células Madre Hematopoyéticas/metabolismo , Adolescente , Adulto , Animales , Biomarcadores , Estudios de Casos y Controles , Recuento de Células , Diferenciación Celular , Niño , Preescolar , Ensayo de Unidades Formadoras de Colonias , Citocinas/metabolismo , Citocinas/farmacología , Modelos Animales de Enfermedad , Supervivencia de Injerto , Enfermedad Granulomatosa Crónica/etiología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Inmunofenotipificación , Mediadores de Inflamación/metabolismo , Ratones , Ratones Transgénicos , Modelos Biológicos , Fenotipo , Transducción de Señal , Adulto Joven
13.
Biomaterials ; 69: 191-200, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26295532

RESUMEN

X-linked chronic granulomatous disease (X-CGD) is an inherited disorder of the immune system. It is characterized by a defect in the production of reactive oxygen species (ROS) in phagocytic cells due to mutations in the NOX2 locus, which encodes gp91phox. Because the success of retroviral gene therapy for X-CGD has been hampered by insertional activation of proto-oncogenes, targeting the insertion of a gp91phox transgene into potential safe harbor sites, such as AAVS1, may represent a valid alternative. To conceptually evaluate this strategy, we generated X-CGD patient-derived induced pluripotent stem cells (iPSCs), which recapitulate the cellular disease phenotype upon granulocytic differentiation. We examined AAVS1-specific zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for their efficacy to target the insertion of a myelo-specific gp91phox cassette to AAVS1. Probably due to their lower cytotoxicity, TALENs were more efficient than ZFNs in generating correctly targeted iPSC colonies, but all corrected iPSC clones showed no signs of mutations at the top-ten predicted off-target sites of both nucleases. Upon differentiation of the corrected X-CGD iPSCs, gp91phox mRNA levels were highly up-regulated and the derived granulocytes exhibited restored ROS production that induced neutrophil extracellular trap (NET) formation. In conclusion, we demonstrate that TALEN-mediated integration of a myelo-specific gp91phox transgene into AAVS1 of patient-derived iPSCs represents a safe and efficient way to generate autologous, functionally corrected granulocytes.


Asunto(s)
Terapia Genética , Granulocitos/metabolismo , Enfermedad Granulomatosa Crónica/genética , Células Madre Pluripotentes Inducidas/metabolismo , Glicoproteínas de Membrana/genética , NADPH Oxidasas/genética , Diferenciación Celular , Línea Celular , Desoxirribonucleasas/genética , Ingeniería Genética , Granulocitos/citología , Enfermedad Granulomatosa Crónica/terapia , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Mieloides/citología , NADPH Oxidasa 2
14.
Curr Gene Ther ; 15(4): 416-27, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25981636

RESUMEN

We report on a series of sequential events leading to long-term survival and cure of pediatric X-linked chronic granulomatous disease (X-CGD) patients after gamma-retroviral gene therapy (GT) and rescue HSCT. Due to therapyrefractory life-threatening infections requiring hematopoietic stem cell transplantation (HSCT) but absence of HLAidentical donors, we treated 2 boys with X-CGD by GT. Following GT both children completely resolved invasive Aspergillus nidulans infections. However, one child developed dual insertional activation of ecotropic viral integration site 1 (EVI1) and signal transducer and activator of transcription 3 (STAT3) genes, leading to myelodysplastic syndrome (MDS) with monosomy 7. Despite resistance to mismatched allo-HSCT with standard myeloablative conditioning, secondary intensified rescue allo-HSCT resulted in 100 % donor chimerism and disappearance of MDS. The other child did not develop MDS despite expansion of a clone with a single insertion in the myelodysplasia syndrome 1 (MDS1) gene and was cured by early standard allo-HSCT. The slowly developing dominance of clones harboring integrations in MDS1-EVI1 may guide clinical intervention strategies, i.e. early rescue allo-HSCT, prior to malignant transformation. GT was essential for both children to survive and to clear therapy-refractory infections, and future GT with safer lentiviral self-inactivated (SIN) vectors may offer a therapeutic alternative for X-CGD patients suffering from life-threatening infections and lacking HLA-identical HSC donors.


Asunto(s)
Terapia Genética/métodos , Enfermedad Granulomatosa Crónica/terapia , Trasplante de Células Madre Hematopoyéticas/métodos , Aspergilosis/terapia , Aspergillus nidulans/patogenicidad , Niño , Deleción Cromosómica , Cromosomas Humanos Par 7 , Proteínas de Unión al ADN/genética , Gammaretrovirus/genética , Terapia Genética/efectos adversos , Humanos , Proteína del Locus del Complejo MDS1 y EV11 , Masculino , Glicoproteínas de Membrana/genética , Síndromes Mielodisplásicos/etiología , NADPH Oxidasa 2 , NADPH Oxidasas/genética , Proto-Oncogenes/genética , Factor de Transcripción STAT3/genética , Factores de Transcripción/genética
15.
Curr Gene Ther ; 14(6): 447-60, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25245086

RESUMEN

Several Phase I/II clinical trials aiming at the correction of X-linked CGD by gene transfer into hematopoietic stem cells (HSCs) have demonstrated the therapeutic potential of gene modified autologous HSCs for the treatment of CGD. Resolution of therapy-resistant bacterial and fungal infections in liver, lung and spinal canal of CGD patients were clearly documented in all trials. However, clinical benefits were not sustained over time due to the failure of gene transduced cells to engraft long-term. Moreover, severe adverse effects were observed in some of the treated patients due to insertional mutagenesis leading to the activation of growth promoting genes and to myeloid malignancy. These setbacks fostered the development of novel safety and efficacy improved vectors that have already entered or are about to enter the clinics. Meanwhile, ongoing research is constantly refining the CGD disease phenotype, including the definition of factors that may explain the unique engraftment phenotype observed in CGD gene therapy trials. This review provides a condensed overview on the current knowledge of the molecular pathomechanisms and clinical manifestations of CGD and summarizes the lessons learned from clinical gene therapy trials, the preclinical progress in vector design and the future perspectives for the gene therapy of CGD.


Asunto(s)
Terapia Genética , Vectores Genéticos/uso terapéutico , Enfermedad Granulomatosa Crónica/genética , Enfermedad Granulomatosa Crónica/terapia , Animales , Humanos
16.
Hum Gene Ther Methods ; 24(3): 151-9, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23489116

RESUMEN

Targeting transgene expression to specific hematopoietic cell lineages could contribute to the safety of retroviral vectors in gene therapeutic applications. Chronic granulomatous disease (CGD), a defect of phagocytic cells, can be managed by gene therapy, using retroviral vectors with targeted expression to myeloid cells. In this context, we analyzed the myelospecificity of the human miR223 promoter, which is known to be strongly upregulated during myeloid differentiation, to drive myeloid-restricted expression of p47(phox) and gp91(phox) in mouse models of CGD and in primary patient-derived cells. The miR223 promoter restricted the expression of p47(phox), gp91(phox), and green fluorescent protein (GFP) within self-inactivating (SIN) gamma- and lentiviral vectors to granulocytes and macrophages, with only marginal expression in lymphocytes or hematopoietic stem and progenitor cells. Furthermore, gene transfer into primary CD34+ cells derived from a p47(phox) patient followed by ex vivo differentiation to neutrophils resulted in restoration of Escherichia coli killing activity by miR223 promoter-mediated p47(phox) expression. These results indicate that the miR223 promoter as an internal promoter within SIN gene therapy vectors is able to efficiently correct the CGD phenotype with negligible activity in hematopoietic progenitors, thereby limiting the risk of insertional oncogenesis and development of clonal dominance.


Asunto(s)
Terapia Genética/métodos , Enfermedad Granulomatosa Crónica/terapia , MicroARNs/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Escherichia coli/aislamiento & purificación , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Granulocitos/inmunología , Granulocitos/metabolismo , Granulocitos/microbiología , Enfermedad Granulomatosa Crónica/metabolismo , Enfermedad Granulomatosa Crónica/patología , Humanos , Lentivirus/genética , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , MicroARNs/genética , NADPH Oxidasa 2 , NADPH Oxidasas/deficiencia , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Fagocitosis , Regiones Promotoras Genéticas
17.
Stem Cells ; 30(4): 599-611, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22311747

RESUMEN

Chronic granulomatous disease (CGD) is an inherited disorder of phagocytes in which NADPH oxidase is defective in generating reactive oxygen species. In this study, we reprogrammed three normal unrelated patient's fibroblasts (p47(phox) and gp91(phox) ) to pluripotency by lentiviral transduction with defined pluripotency factors. These induced pluripotent stem cells (iPSC) share the morphological features of human embryonic stem cells, express the key pluripotency factors, and possess high telomerase activity. Furthermore, all the iPSC lines formed embryoid bodies in vitro containing cells originating from all three germ layers and were capable of teratoma formation in vivo. They were isogenic with the original patient fibroblasts, exhibited normal karyotype, and retained the p47(phox) or gp91(pho) (x) mutations found in the patient fibroblasts. We further demonstrated that these iPSC could be differentiated into monocytes and macrophages with a similar cytokine profile to blood-derived macrophages under resting conditions. Most importantly, CGD-patient-specific iPSC-derived macrophages showed normal phagocytic properties but lacked reactive oxygen species production, which correlates with clinical diagnosis of CGD in the patients. Together these results suggest that CGD-patient-specific iPSC lines represent an important tool for modeling CGD disease phenotypes, screening candidate drugs, and the development of gene therapy.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Enfermedad Granulomatosa Crónica/patología , Células Madre Pluripotentes Inducidas/patología , Modelos Biológicos , Diferenciación Celular , Línea Celular , Citocinas/metabolismo , Humanos , Cariotipificación , Cinética , Macrófagos/citología , Macrófagos/metabolismo , Monocitos/citología , Monocitos/metabolismo , NADPH Oxidasas/metabolismo , Fenotipo , Especies Reactivas de Oxígeno/metabolismo
18.
J Allergy Clin Immunol ; 127(5): 1243-52.e7, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21376380

RESUMEN

BACKGROUND: Aspergillus spp infection is a potentially lethal disease in patients with neutropenia or impaired neutrophil function. We showed previously that Aspergillus hyphae, too large for neutrophil phagocytosis, are inhibited by reactive oxygen species-dependent neutrophil extracellular trap (NET) formation. This process is defective in chronic granulomatous disease (CGD) because of impaired phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase function. OBJECTIVE: To determine the antifungal agent and mechanism responsible for reconstitution of Aspergillus growth inhibition within NETs after complementation of NADPH oxidase function by gene therapy (GT) for CGD. METHODS: Antifungal activity of free and NET-released calprotectin was assessed by incubation of Aspergillus nidulans with purified calprotectin, induced NETs from human controls, and CGD neutrophils after GT in the presence or absence of Zn(2+) or α-S100A9 antibody, and with induced NETs from wild-type or S100A9(-/-) mouse neutrophils. RESULTS: We identified the host Zn(2+) chelator calprotectin as a neutrophil-associated antifungal agent expressed within NETs, reversibly preventing A nidulans growth at low concentrations, and leading to irreversible fungal starvation at higher concentrations. Specific antibody-blocking and Zn(2+) addition abolished calprotectin-mediated inhibition of A nidulans proliferation in vitro. The role of calprotectin in anti-Aspergillus defense was confirmed in calprotectin knockout mice. CONCLUSION: Reconstituted NET formation by GT for human CGD was associated with rapid cure of pre-existing therapy-refractory invasive pulmonary aspergillosis in vivo, underlining the role of functional NADPH oxidase in NET formation and calprotectin release for antifungal activity. These results demonstrate the critical role of calprotectin in human innate immune defense against Aspergillus infection.


Asunto(s)
Aspergilosis/inmunología , Aspergillus nidulans/efectos de los fármacos , Terapia Genética/métodos , Enfermedad Granulomatosa Crónica/terapia , Complejo de Antígeno L1 de Leucocito/metabolismo , Neutrófilos/inmunología , Animales , Antifúngicos/metabolismo , Antifúngicos/farmacología , Aspergilosis/microbiología , Niño , Enfermedad Granulomatosa Crónica/complicaciones , Enfermedad Granulomatosa Crónica/inmunología , Humanos , Complejo de Antígeno L1 de Leucocito/farmacología , Masculino , Ratones , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Neutrófilos/metabolismo , Neutrófilos/fisiología , Especies Reactivas de Oxígeno/metabolismo
19.
Clin Infect Dis ; 49(11): 1703-10, 2009 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-19874205

RESUMEN

BACKGROUND: Chronic granulomatous disease (CGD) is a rare inherited disease of the phagocyte NADPH oxidase system that causes defective production of toxic oxygen metabolites, impaired bacterial and fungal killing, and recurrent life-threatening infections, mostly by catalase-producing organisms. We report for the first time, to our knowledge, chronic infections with Actinomyces species in 10 patients with CGD. Actinomycosis is a chronic granulomatous condition that commonly manifests as cervicofacial, pulmonary, or abdominal disease, caused by slowly progressive infection with oral and gastrointestinal commensal Actinomyces species. Treatment of actinomycosis is usually simple in immunocompetent individuals, requiring long-term, high-dose intravenous penicillin, but is more complicated in those with CGD because of delayed diagnosis and an increased risk of chronic invasive or debilitating disease. METHODS: Actinomyces was identified by culture, staining, 16S ribosomal DNA polymerase chain reaction, and/or a complement fixation test in 10 patients with CGD. RESULTS: All 10 patients presented with a history of fever and elevated inflammatory signs without evident focus. Diagnosis was delayed and clinical course severe and protracted despite high-dose intravenous antibiotic therapy and/or surgery. These results suggest an unrecognized and unanticipated susceptibility to weakly pathogenic Actinomyces species in patients with CGD because these are catalase-negative organisms previously thought to be nonpathogenic in CGD. CONCLUSIONS: Actinomycosis should be vigorously sought and promptly treated in patients with CGD presenting with uncommon and prolonged clinical signs of infection. Actinomycosis is a catalase-negative infection important to consider in CGD.


Asunto(s)
Actinomyces/patogenicidad , Actinomicosis/diagnóstico , Actinomicosis/tratamiento farmacológico , Enfermedades Transmisibles Emergentes/microbiología , Enfermedades Transmisibles Emergentes/patología , Enfermedad Granulomatosa Crónica/microbiología , Actinomyces/genética , Actinomicosis/cirugía , Actinomicosis/terapia , Adolescente , Adulto , Amoxicilina/uso terapéutico , Antibacterianos/uso terapéutico , Azitromicina/uso terapéutico , Trasplante de Médula Ósea , Ceftriaxona/uso terapéutico , Niño , Clindamicina/uso terapéutico , ADN Ribosómico/genética , Femenino , Humanos , Masculino , Meropenem , Penicilina G/uso terapéutico , Penicilina V/uso terapéutico , Reacción en Cadena de la Polimerasa , Sulfametoxazol/uso terapéutico , Tienamicinas/uso terapéutico , Trimetoprim/uso terapéutico , Adulto Joven
20.
Blood ; 114(13): 2619-22, 2009 Sep 24.
Artículo en Inglés | MEDLINE | ID: mdl-19541821

RESUMEN

Chronic granulomatous disease (CGD) patients have impaired nicotinamide adenine dinucleotide phosphate (NADPH) oxidase function, resulting in poor antimicrobial activity of neutrophils, including the inability to generate neutrophil extracellular traps (NETs). Invasive aspergillosis is the leading cause of death in patients with CGD; it is unclear how neutrophils control Aspergillus species in healthy persons. The aim of this study was to determine whether gene therapy restores NET formation in CGD by complementation of NADPH oxidase function, and whether NETs have antimicrobial activity against Aspergillus nidulans. Here we show that reconstitution of NET formation by gene therapy in a patient with CGD restores neutrophil elimination of A nidulans conidia and hyphae and is associated with rapid cure of preexisting therapy refractory invasive pulmonary aspergillosis, underlining the role of functional NADPH oxidase in NET formation and antifungal activity.


Asunto(s)
Aspergilosis/prevención & control , Quimiotaxis de Leucocito/genética , Terapia Genética , Enfermedad Granulomatosa Crónica/terapia , Neutrófilos/fisiología , Antifúngicos/metabolismo , Aspergilosis/etiología , Aspergilosis/genética , Aspergilosis/metabolismo , Aspergillus nidulans , Quimiotaxis de Leucocito/inmunología , Niño , Terapia Genética/métodos , Enfermedad Granulomatosa Crónica/complicaciones , Enfermedad Granulomatosa Crónica/genética , Enfermedad Granulomatosa Crónica/inmunología , Humanos , Masculino , Neutrófilos/metabolismo
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