Chronic granulomatous disease (CGD) is a group of rare genetic immunodeficiencies (total incidence 1:70’000 newborns) caused by mutations in the NADPH oxidase, a protein complex catalysing the destruction of ingested microorganisms within specialised immune cells called phagocytes. Affected patients suffer from severe recurrent bacterial and fungal infections of body surfaces and internal organs. We have previously shown that defective formation of neutrophil extracellular traps (NETs, i.e. net-like DNA structures which bind and trap microbes and kill them by concentrated anti-microbial proteins) is responsible for severe fungal and bacterial infection in CGD. The disease is also characterised by abnormally exuberant inflammatory responses leading to severe bowel inflammation, genitourinary obstruction, and poor wound healing. We have shown that activation of proinflammatory caspase-1 and IL-1 secretion are elevated in CGD phagocytes, especially in cells derived from patients with inflammatory complications. A dual role for NADPH oxidase in control of infections and of inflammation is thus likely.

Macroautophagy is a cell-protective process that sequesters cytoplasmic material and delivers it to lysosomes for degradation and recycling. In addition, it has recently been implied in the innate immune response to extracellular pathogens after phagocytosis in an NADPH oxidase dependent way, in ER and Golgi apparatus independent exocytosis and in the control of the inflammasome. Defective inflammasome control has been described to lead to caspase-1 activation and IL-1 secretion in a set of rare autoinflammatory diseases. Mutations in genes involved in macroautophagy have been found associated with Morbus Crohn, an inflammatory bowel disease which clinically and pathologically strongly resembles CGD bowel inflammation. Taken together, defective macroautophagy may represent the missing link between the two main features of CGD: overwhelming infection and exuberant inflammation.

The project aims first at unravelling the role of macroautophagy in the CGD disease process with regard to NADPH oxidase function in two types of phagocytes – neutrophils, which are first line of defence against infections, and macrophages which limit inflammatory reactions. The second objective of the proposal is identification of new therapeutic targets, and efficient correction of defective immune defence and hyperinflammation by novel gene therapy (GT) approaches. The previous results of our group on GT for CGD have shown that a GT approach for treatment of this devastating disease is feasible and may substantially contribute to improve patient’s health. Novel safer gene transfer systems (SIN-vectors conferring expression limited to phagocytes vs. targeted insertion into safe genomic harbors by the Zinc-finger nuclease technology) will be compared with regard to optimal control of infections and inflammation. CGD patient derived induced pluripotent stem cells (iPSC) will be used for that purpose. Altogether this laboratory model could serve as definitive proof of functional efficiency and safety of defined GT vectors before clinical use of the vectors in future human gene therapy trials for CGD.

CGD remains a lethal disease with a median survival of 30 years despite lifelong prophylactic antibiotic and antimycotic treatment. New approaches to causative treatment are urgently needed. This project supported by GEBERT RÜF STIFTUNG will help to characterise the so far poorly understood disease pathophysiology of the rare genetic immunodeficiency CGD. Analysis of macroautophagy presents a new approach towards elucidating CGD biochemical disease processes with regard to defective anti-microbial defence by NETs and exuberant inflammation. By implementing the novel iPSC engineering technology, genetic correction of the NADPH oxidase and a novel biological defect (ROS dependent macroautophagy) in vitro will allow for the first time to predict clinical efficiency and safety of gene therapy by different novel gene transfer systems for a defined patient in a laboratory model thus granting direct transfer of basic research findings into clinical practice. The project is an innovative interdisciplinary approach between paediatric clinical immunology, basic immunology, cell biology and molecular biology, meeting high scientific and technological standards.

The project has generally been a success, leading to several publications and submitted manuscripts. More importantly it has led to identification of a new gene therapy vector for the treatment of patients with CGD, conferring optimal expression and function of NADPH oxidase in phagocytes. We generated and characterized four iPSC lines from CGD patients (Jiang et al. Stem Cells 2012). Two more iPSC cell lines are currently established, but have not yet been published. Using this iPSC model, we could show that this vector can reconstitute defective immune defence; it will now enter final pre-clinical testing for future clinical phase I/II gene therapy trials for patients with CGD. Analysis of macroautophagy has shown a differential role of this process in phagocyte subtypes: In CGD neutrophils classical macroautophagy levels were not different to control cells. However in CGD macrophages recruitment of Atg8/LC3 to the membranes of phagosomes was absent, leading to reduced MHC class II fungal antigen presentation. This could explain in part the susceptibility of CGD patients to fungal infection (Romao et al J Cell Biol 2013). Based on the results from this Gebert Rüf Stiftung funded project, further start-up funding has been acquired for separate MD-PhD and PhD.

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Dreyer A.K., Hoffmann D., Lachmann N., Ackermann M., Steinemann D., Timm B., Siler U., Reichenbach J., Grez M., Moritz T., Schambach A., CathomenT. TALEN mediated functional correction of X-linked chronic granulomatous disease in patient derived induced pluripotent stem cells. Biomaterials 2015; 69: 191-200;
Siler U †, Paruzynski A †, Holtgreve-Grez H, Kuzmenko E, Koehl U, Renner ED, Alhan C, van de Loosdrecht AA, Schwäble J, Pfluger T, Tchinda J, Schmugge M, Jauch A, Naundorf S, Kühlcke K, Notheis G, Güngör T, v. Kalle C, Schmidt M +, Grez M +, Seger R +, Reichenbach J. Successful Combination of Sequential Gene Therapy and Rescue allo-HSCT in two Children with X-CGD - importance of timing. Current Gene Therapy, 2015;15: 416-27, †, + These authors contributed equally to the work;
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Romao S et al. Autophagy proteins stabilize pathogen-containing phagosomes for prolonged MHC II antigen processing. J Cell Biol 2013;203(5):757-66;
Brendel C, et al. Human miR223 Promoter as Novel Myelospecific Promoter for CGD Gene Therapy. Human Gene Ther 2013; 24(3):151-9;
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Bianchi M et al. Restoration of anti-Aspergillus defense by NETs in human CGD is Calprotectin dependent. J Allergy Clin Immunol 2011;127(5):1243-1252.

Wirkung neuer Gentherapien effizienter prüfen, UZH Medienmitteilung, 21.3.2017
Auf der Suche nach der perfekten Genfähre€, UZH News, 26.6.2012