title: Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells.
abstract: Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency due to dysfunction of the phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex leading to severe and recurrent infections in early childhood. The main genetic form is the X-linked CGD leading to the absence of cytochromeb558composed of NOX2 and p22phox, the membrane partners of the NADPH oxidase complex. The first cause of death of CGD patients is pulmonary infections. Recombinant proteoliposome-based therapy is an emerging and innovative approach for membrane protein delivery, which could be an alternative local, targeted treatment to fight lung infections in CGD patients. We developed an enzyme therapy using recombinant NOX2/p22phoxliposomes to supply the NADPH oxidase activity in X0-linked CGD (X0-CGD) macrophages. Using an optimized prokaryotic cell-free protein synthesis system, a recombinant cytochromeb558containing functional hemes was produced and directly inserted into the lipid bilayer of specific liposomes. The size of the NOX2/p22phoxliposomes was estimated to be around 700 nm. These proteoliposomes were able to generate reactive oxygen species (ROS) in an activated reconstituted cell-free NADPH oxidase activation assay in the presence of recombinant p47phox, p67phoxand Rac, the cytosolic components of the NADPH oxidase complex. Furthermore, using flow cytometry and fluorescence microscopy, we demonstrated that cytochromeb558was successfully delivered to the plasma membrane of X0-CGD-induced pluripotent stem cell (iPSC)-derived macrophages. In addition, NADPH oxidase activity was restored in X0-CGD iPSC-derived macrophages treated with NOX2/p22phoxliposomes for 8 h without any toxicity. In conclusion, we confirmed that proteoliposomes provide a new promising technology for the delivery of functional proteins to the membrane of targeted cells. This efficient liposomal enzyme replacement therapy will be useful for future treatment of pulmonary infections in CGD patients refractory to conventional anti-infectious treatments.
authors.affiliation: UMR CNRS 5525, University of Grenoble Alpes, Grenoble, France; CGD Diagnosis and Research Centre, University Hospital Centre of Grenoble Alpes, Grenoble, France.
authors.forename: Julie
authors.initials: J
authors.lastname: Brault
authors.affiliation: Synthelis SAS, La Tronche, France.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Vaganay
authors.affiliation: IBS, University of Grenoble Alpes, Grenoble, France; CNRS, IBS, University Grenoble Alpes, Grenoble, France; CEA, IBS, University of Grenoble Alpes, Grenoble, France.
authors.forename: Aline
authors.initials: A
authors.lastname: Le Roy
authors.affiliation: UMR CNRS 5525, University of Grenoble Alpes, Grenoble, France.
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Lenormand
authors.affiliation: Synthelis SAS, La Tronche, France.
authors.forename: Sandra
authors.initials: S
authors.lastname: Cortes
authors.affiliation: UMR CNRS 5525, University of Grenoble Alpes, Grenoble, France; CGD Diagnosis and Research Centre, University Hospital Centre of Grenoble Alpes, Grenoble, France.
authors.forename: Marie José
authors.initials: MJ
authors.lastname: Stasia
mesh_terms: D002175:Candida / Q000378:metabolism; D002454:Cell Differentiation; D002462:Cell Membrane / Q000378:metabolism; D002675:Child, Preschool; D040181:Genetic Diseases, X-Linked / Q000188:drug therapy*; D006105:Granulomatous Disease, Chronic / Q000188:drug therapy*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000473:pathology*; D008264:Macrophages / Q000378:metabolism / Q000473:pathology*; D019255:NADPH Oxidases / Q000378:metabolism; D010587:Phagocytosis; D021122:Protein Subunits / Q000378:metabolism; D011510:Proteolipids / Q000627:therapeutic use*; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D021122:Protein Subunits; D011510:Proteolipids; D017382:Reactive Oxygen Species; C020485:proteoliposomes; D019255:NADPH Oxidases
keywords: NADPH oxidase; chronic granulomatous disease; induced pluripotent stem cells; macrophages; protein therapy; proteoliposomes