Mouse model of X–linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production

Pollock, Jonathan; Williams, David A.; Gifford, Mary; Li, Ling Lin; Du, Xunxiang; Fisherman, Jason S.; Orkin, Stuart H.; Doerschuk, Claire M.; Dinauer, Mary C.

doi:10.1038/ng0295-202

Cited by 848 publications

(820 citation statements)

References 34 publications

Supporting

Mentioning

779

Contrasting

Unclassified

Order By: Relevance

“…Nox2 −/− mice on a C57BL/6J background (Pollock et al, 1995), originally obtained from Jackson Laboratories (Bar Harbour, USA), were bred from an established colony at Queen's University Belfast. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al , 2010; McGrath and Lilley, 2015).…”

Section: Methodsmentioning

confidence: 99%

Signalling mechanisms underlying doxorubicin and Nox2 NADPH oxidase‐induced cardiomyopathy: involvement of mitofusin‐2

McLaughlin

Zhao

O’Neill

et al. 2017

British J Pharmacology

View full text Add to dashboard Cite

Background and PurposeThe anthracycline doxorubicin (DOX), although successful as a first‐line cancer treatment, induces cardiotoxicity linked with increased production of myocardial ROS, with Nox2 NADPH oxidase‐derived superoxide reported to play a key role. The aim of this study was to identify novel mechanisms underlying development of cardiac remodelling/dysfunction further to DOX‐stimulated Nox2 activation.Experimental ApproachNox2−/− and wild‐type (WT) littermate mice were administered DOX (12 mg·kg−1 over 3 weeks) prior to study at 4 weeks. Detailed mechanisms were investigated in murine HL‐1 cardiomyocytes, employing a robust model of oxidative stress, gene silencing and pharmacological tools.Key ResultsDOX‐induced cardiac dysfunction, cardiomyocyte remodelling, superoxide production and apoptosis in WT mice were attenuated in Nox2−/− mice. Transcriptional analysis of left ventricular tissue identified 152 differentially regulated genes (using adjusted P < 0.1) in DOX‐treated Nox2−/− versus WT mice, and network analysis highlighted ‘Cell death and survival’ as the biological function most significant to the dataset. The mitochondrial membrane protein, mitofusin‐2 (Mfn2), appeared as a strong candidate, with increased expression (1.5‐fold), confirmed by qPCR (1.3‐fold), matching clear published evidence of promotion of cardiomyocyte cell death. In HL‐1 cardiomyocytes, targeted siRNA knockdown of Nox2 decreased Mfn2 protein expression, but not vice versa. While inhibition of Nox2 activity along with DOX treatment attenuated its apoptotic and cytotoxic effects, reduced apoptosis after Mfn2 silencing reflected a sustained cytotoxic response and reduced cell viability.Conclusions and ImplicationsDOX‐induced and Nox2‐mediated up‐regulation of Mfn2, rather than contributing to cardiomyocyte dysfunction through apoptotic pathways, appears to promote a protective mechanism.Linked ArticlesThis article is part of a themed section on New Insights into Cardiotoxicity Caused by Chemotherapeutic Agents. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.21/issuetoc

show abstract

Section: Methodsmentioning

confidence: 99%

Signalling mechanisms underlying doxorubicin and Nox2 NADPH oxidase‐induced cardiomyopathy: involvement of mitofusin‐2

McLaughlin

Zhao

O’Neill

et al. 2017

British J Pharmacology

View full text Add to dashboard Cite

show abstract

“…Murine Lin À cells were isolated from bone marrow samples harvested from the femurs and tibias of B6.129S6-Cybb tm1Din/J mice, 49,50 (Ly5.2) using the Miltenyi Lineage Cell Depletion Kit (Miltenyi, Bergisch Gladbach, Germany) following the manufacturer's instructions. Lin À cells were cultured in StemSpan serum-free medium (StemCell Technologies, Grenoble, France) supplemented with 1% penicillin/streptomycin and cytokines.…”

Section: Cell Culturementioning

confidence: 99%

Physiological regulation of transgene expression by a lentiviral vector containing the A2UCOE linked to a myeloid promoter

et al. 2011

View full text Add to dashboard Cite

Protection against epigenetic silencing is a desirable feature of future gene therapy vectors, in particular for those applications in which transgene expression will not confer growth advantage to gene-transduced cells. The ubiquitous chromatin opening element (UCOE) consisting of the methylation-free CpG island encompassing the dual divergently transcribed promoters of the human HNRPA2B1-CBX3 housekeeping genes (A2UCOE) has been shown to shield constitutive active heterologous promoters from epigenetic modifications and chromosomal position effects. However, it is unclear if this element can be used to improve expression from tissue-specific enhancer/promoters, while maintaining tissue specificity in hematopoietic cells. Here, we evaluated the potential of the A2UCOE in combination with the myeloid-specific myeloid related protein 8 (MRP8) promoter to target transgene expression specifically to myeloid cells in vitro and in vivo from a self-inactivating lentiviral vector. The inclusion of the A2UCOE did not interfere with specific upregulation of MRP8 promoter activity during myeloid differentiation and mediated sustained and vector copy-dependent expression in myeloid cells. Notably, the A2UCOE did not protect the MRP8 promoter from methylation in the P19 embryonal carcinoma cell line, suggesting that this element maintains the inherent epigenetic state and transcriptional activity of cellular promoters in their native configuration. Thus, the A2UCOE could represent a useful protective genetic element in gene therapy vectors, ensuring physiological transcriptional regulation of tissue-specific promoters independent of the chromosomal integration site.

show abstract

“…HSC from gp91 knockout male mice were transduced ex vivo and transplanted into gp91 knockout females. 20 Chimerism and proviral copy numbers were first determined at 5 weeks posttransplantation in the peripheral blood of the recipient animals and remained stable even at 6 months after the graft. At 8 weeks posttransplantation, levels of NADPH enzymatic activity were measured using the standard dihydrorhodamine (DHR) test.…”

Section: In Vivo Expression Of Most Promising Candidatesmentioning

confidence: 99%

Lineage- and stage-restricted lentiviral vectors for the gene therapy of chronic granulomatous disease

et al. 2011

View full text Add to dashboard Cite

Insertional mutagenesis represents a serious adverse effect of gene therapy with integrating vectors. However, although uncontrolled activation of growth-promoting genes in stem cells can predictably lead to oncological processes, this is far less likely if vector transcriptional activity can be restricted to fully differentiated cells. Diseases requiring phenotypic correction only in mature cells offer such an opportunity, provided that lineage/stage-restricted systems can be properly tailored. In this study, we followed this reasoning to design lentiviral vectors for the gene therapy of chronic granulomatous disease (CGD), an immune deficiency due a loss of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes, most often secondary to mutations in gp91 phox . Using self-inactivating HIV1-derived vectors as background, we first expressed enhanced green fluorescent protein (eGFP) from a minimal gp91 phox promoter, adding various natural or synthetic transcriptional regulatory elements to foster both specificity and potency. The resulting vectors were assessed either by transplantation or by lentiviral transgenesis, searching for combinations conferring strong and specific expression into mature phagocytic cells. The most promising vector was modified to express gp91 phox and used to treat CGD mice. High-level restoration of NADPH activity was documented in granulocytes from the treated animals. We propose that this lineage-specific lentiviral vector is a suitable candidate for the gene therapy of CGD.

show abstract

Mouse model of X–linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production

Cited by 848 publications

References 34 publications

Signalling mechanisms underlying doxorubicin and Nox2 NADPH oxidase‐induced cardiomyopathy: involvement of mitofusin‐2

Signalling mechanisms underlying doxorubicin and Nox2 NADPH oxidase‐induced cardiomyopathy: involvement of mitofusin‐2

Physiological regulation of transgene expression by a lentiviral vector containing the A2UCOE linked to a myeloid promoter

Lineage- and stage-restricted lentiviral vectors for the gene therapy of chronic granulomatous disease

Contact Info

Product

Resources

About