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.

A Novel Mutation in the NCF2 Gene in a CGD Patient With Chronic Recurrent Pneumopathy.

Chronic granulomatous disease (CGD) is an inherited, genetically heterogeneous disease characterized by defective phagocytic cell microbicidal function, leading to increased susceptibility to bacterial and fungal infections. CGD is caused by mutations in components of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system, which is responsible for reactive oxygen species production during phagocytosis. Mutations in the neutrophil cytosolic factor 2 (NCF2) gene account for <5% of all cases. Here, we report a case of a 2-year-old female with persistent recurrent pneumopathy, even under trimethoprim-sulfamethoxazole (TMP-SMX) and itraconazole prophylaxis combined with IFNγ treatment. Genetic analysis revealed a novel homozygous mutation in NCF2, sequence depletion in a splicing region (c.256_257+2delAAGT NM_000433), leading to a K86Ifs*2 residue change in the p67-phox protein.

Papel do fator nuclear kappa B (NF-κB) na expressâo do gene NCF1 em leucócitos de indivíduos normais, e pacientes com doença granulomatosa crônica, displasia ectodérmica anidrótica, ou com defeitos no eixo IL-12/23-IFN-γ / Role of nuclear factor kappa B (NF-κB) on NCF1 gene expression in leukocytes from normal individuals, and patients with chronic granulomatous disease, anhidrotic ectodermal dysplasia, or with defects in the IL-12/23/IFN-γ axis

Objetivo: Analisamos a relevância do NF-κB sobre a expressão do gene NCF1 em células mielóides U937 selvagens (U937) ou transfectadas com um repressor do NF-κB (IκBα-S32A/S36A - U937 IκBα-S32A/S36A) ou transfectadas com o vetor vazio (U937 pCMV3) e em células B imortalizadas pelo vírus Epstein-Barr (EBV) de pacientes com displasia ectodérmica anidrótica com imunodeficiência (EDA-ID) ou com doença granulomatosa crônica (CGD) devido a mutações no gene NCF1, ou de pacientes portadores de defeitos do eixo IL-12/ 23-IFN-γ. Métodos: O RNA celular total foi isolado pelo método TRI-zol®. Os cDNAs foram produzidos utilizando-se o SuperScript™ III e amplificados por Real-time PCR (SYBR® Green Master Mix). Resultados: Células U937 IKBα-S32A/S36A mostraram significante decréscimo na expressão do gene NCF1 comparadas com as células U937. A expressão do gene NCF1 em células EDA-ID S32I foi significativamente menor que em controles saudáveis, assim como em células EDA-ID NEMO/IKKγ X420W na mesma comparação. Estes resultados foram similares aos encontrados em células de pacientes CGD devido à mutação autossômica recessiva no gene NCF1 quando comparados com o controle normal. Defeitos nos receptores IFNGR1 e IFNGR2 levam à diminuição da expressão do gene NCF1 (p<0,05, Mann Whitney). Conclusões: Estes resultados mostram que o NF-κB é necessário para a expressão do gene NCF1, que possivelmente as subunidades p50 e/ou p65 do NF-κB ligam-se funcionalmente à região "upstream" do gene NCF1 e que defeitos no eixo IL-12/ 23-IFN-γ influenciam a expressão do gene NCF1.

Objective: We analyzed the relevance of NF-κB on NCF1 gene expression in regular myeloid U937 cells (U937), or transfected with a NF-κB repressor (IκBo-S32A/S36A - U937 IκBo-S32A/S36A), or transfected with the empty vector (U937 pCMV3), and in B cells immortalized by Epstein-Barr vírus (EBV) from patients with anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID), or with chronic granulomatous desease (CGD) due to mutations in NCF1 gene, or from patients with IL-12/23-IFN-γ axis defects. Methods: Total RNA was isolated by the TRIzol® method. The cDNAs were produced by Super Script™ III and amplified by Real-time PCR (SYBR Green Master Mix). Results: U937 IκBo-S32A/S36A cells showed significant decrease in the NCF1 gene expression compared with the U937 cells. The NCF1 gene expression was significantly lower in EDA-ID S32I cells compared to healthy controls as well as in EDA-ID NEMO/IKKy X420W cells. These results were similar to those obtained in the CGD patient cells due recessive mutations in the NCF1 gene compared to healthy controls. Cells form patients with defects in IFNGR1 e IFNGR2 receptors also presented decreased NCF1 gene expression (p<0,05, Mann Whitney). Conclusion: These results show that the NF-κB is necessary for NCF1 expression, possibly the p50 and/or p65 NF-κB subunits bind functionally to the upstream region of the NCF1 gene and that IL-12/23-IFN-γ axis defects also influence NCF1 gene expression.

Essential role of nuclear factor-kappaB for NADPH oxidase activity in normal and anhidrotic ectodermal dysplasia leukocytes.

This work investigated the functional role of nuclear factor-kappaB (NF-kappaB) in respiratory burst activity and in expression of the human phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase genes CYBB, CYBA, NCF1, and NCF2. U937 cells with a stably transfected repressor of NF-kappaB (IkappaBalpha-S32A/S36A) demonstrated significantly lower superoxide release and lower CYBB and NCF1 gene expression compared with control U937 cells. We further tested Epstein-Barr virus (EBV)-transformed B cells from patients with anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID), an inherited disorder of NF-kappaB function. Superoxide release and CYBB gene expression by EDA-ID cells were significantly decreased compared with healthy cells and similar to cells from patients with X-linked chronic granulomatous disease (X91(0) CGD). NCF1 gene expression in EDA-ID S32I cells was decreased compared with healthy control cells and similar to that in autosomal recessive (A47(0)) CGD cells. Gel shift assays demonstrated loss of recombinant human p50 binding to a NF-kappaB site 5' to the CYBB gene in U937 cells treated with NF-kappaB inhibitors, repressor-transfected U937 cells, and EDA-ID patients' cells. Zymosan phagocytosis was not affected by transfection of U937 cells with the NF-kappaB repressor. These studies show that NF-kappaB is necessary for CYBB and NCF1 gene expression and activation of the phagocyte NADPH oxidase in this model system.