Gene therapy enhances deoxyribonuclease I treatment in antimyeloperoxidase glomerulonephritis
Anne Cao Le, Virginie Oudin, Jonathan Dick, Maliha A. Alikhan, Timothy A. Gottschalk, Lu Lu, Kate E. Lawlor, Daniel Koo Yuk Cheong, Mawj Mandwie, Ian E. Alexander, A.R. Kitching, Poh-Yi Gan, Grant J. Logan, Kim M. O’Sullivan
Anne Cao Le, Virginie Oudin, Jonathan Dick, Maliha A. Alikhan, Timothy A. Gottschalk, Lu Lu, Kate E. Lawlor, Daniel Koo Yuk Cheong, Mawj Mandwie, Ian E. Alexander, A.R. Kitching, Poh-Yi Gan, Grant J. Logan, Kim M. O’Sullivan
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Research Article Inflammation

Gene therapy enhances deoxyribonuclease I treatment in antimyeloperoxidase glomerulonephritis

Abstract

Extracellular DNA (ecDNA) released from injured and dying cells powerfully induces injurious inflammation. In this study we define the role of ecDNA in systemic vasculitis affecting the kidney, using human kidney biopsies and murine models of myeloperoxidase anti-neutrophil cytoplasmic antibody-associated glomerulonephritis (MPO-ANCA GN). Twice daily administration of intravenous deoxyribonuclease I (ivDNase I) in 2 models of anti-MPO GN reduced glomerular deposition of ecDNA, histological injury, leukocyte infiltration, and NETosis. Comprehensive investigation into DNase I modes of action revealed that after exposure to MPO, DNase I reduced lymph node DC numbers and their activation status, resulting in decreased frequency of MPO-specific CD4+ effector T cells (IFN-γ and IL-17A producing) and reductions in dermal anti-MPO delayed type hypersensitivity responses. To overcome the translational obstacle of the short half-life of DNase I (<5 hours), we tested an adeno-associated viral vector encoding DNase I. This method of DNase I delivery was more effective, as in addition to the histological and antiinflammatory changes described above, a single vector treatment also reduced circulating MPO-ANCA titers and albuminuria. These results indicate ecDNA is a potent driver of anti-MPO GN and DNase I is a potential therapeutic that can be delivered using gene technology.

Authors

Anne Cao Le, Virginie Oudin, Jonathan Dick, Maliha A. Alikhan, Timothy A. Gottschalk, Lu Lu, Kate E. Lawlor, Daniel Koo Yuk Cheong, Mawj Mandwie, Ian E. Alexander, A.R. Kitching, Poh-Yi Gan, Grant J. Logan, Kim M. O’Sullivan

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Figure 10

vec-DNase I treatment reduces kidney injury, glomerular leukocyte infiltration, cell death, and NETosis.

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vec-DNase I treatment reduces kidney injury, glomerular leukocyte infilt...
Treatment with vec-DNase I significantly reduces 24-hour albuminuria (A) and histological glomerular injury compared with the control GFP vector (B). Glomerular leukocyte recruitment is reduced in mice treated with both the exogenous DNase I and vec-DNase I when compared with the control AAV-GFP vector (C–E). Both segmental necrosis and markers of cell death RIPK3 and caspase-3 are reduced in mice treated with the vec-DNase I compared with the control vec-GFP (F and G). NET assays stimulated with PMA and treated with DNase I at 1 μg/mL demonstrate attenuation of NETs, with DNA in blue, MPO in red, H3Cit in green, and PAD4 in gray/blue (H). pHrodo Green S. aureus bioparticles were used for phagocytosis assays with neutrophils incubated with DNase I at increased concentrations 0–4 μg/mL (purple bars) and compared with untreated neutrophils (orange bar) to show that DNase I does not inhibit neutrophil phagocytosis (I). *P < 0.05, **P < 0.01, ***P < 0.001. In vivo animal data are median (IQR) from 8 mice in each group analyzed by Kruskal-Wallis test with Dunn’s post hoc test for multiple comparisons. Dotted line represents OVA-immunized mice as a reference control. In vitro phagocytosis data expressed as the average of duplicate wells with data expressed as the median (IQR). Dotted line represents bead-only reference control. RIPK3, receptor-interacting serine/threonine-protein kinase 3; vec, adeno-associated viral vector; Ctrl, control; DNase I, deoxyribonuclease I; GFP, green fluorescent protein; H3Cit, citrullinated histone 3; IQR, interquartile range; MPO, myeloperoxidase; PAD4, peptidyl arginine deiminase 4; PMA, phorbal myristate acetate.