Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells

Abstract

International audienceChronic 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 cytochrome b558 composed of NOX2 and p22 phox , 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/p22 phox liposomes to supply the NADPH oxidase activity in X0-linked CGD (X0-CGD) macrophages. Using an optimized prokaryotic cell-free protein synthesis system, a recombinant cytochrome b558 containing functional hemes was produced and directly inserted into the lipid bilayer of specific liposomes. The size of the NOX2/p22 phox liposomes 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 p47 phox , p67 phox and Rac, the cytosolic components of the NADPH oxidase complex. Furthermore, using flow cytometry and fluorescence microscopy, we demonstrated that cytochrome b558 was 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/p22 phox liposomes 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

Human Induced Pluripotent Stem Cell–Derived Macrophages for Unraveling Human Macrophage Biology

“…32 Beyond the use of patient-derived IPSDM, CRISPR/Cas9-edited IPSDM for key regulators in inflammation and lipid metabolism pathways can be exploited for the development of novel therapeutics and delivery systems. 41 The development of organoid systems derived from isogenic iPSC lines will allow drug effects to be tested within the context of the symbiotic interactions between macrophages and their host tissue with the potential for development of individualized treatment strategies.…”

Human-induced pluripotent stem cell-derived macrophages and their immunological function in response to tuberculosis infection

“…Among the multiple lineages of differentiated cells derived from hiPS, macrophages hold great promise in disease pathogenesis or therapy, such as cancer [ 29 , 30 ], Mendelian disease [ 31 ], Alzheimer’s disease [ 30 ], HIV [ 32 ], Chlamydia trachomatis pathogenesis [ 33 ], chronic granulomatous disease [ 34 ], and X-linked chronic granulomatous diseases [ 35 ], particularly tuberculosis. Despite substantial appreciation for the dual function of macrophages in innate immunity and lipid metabolism, understanding human macrophage biology has been hampered by the lack of reliable and scalable models for cellular and genetic studies.…”

“…Furthermore, the generation of hiPS-derived macrophages focuses on the application of therapy or the pathogenesis of cancer [ 29 , 30 ], Mendelian disease [ 31 ], Alzheimer’s disease [ 30 ], HIV [ 32 ], Chlamydia trachomatis infections [ 33 ], chronic granulomatous disease [ 34 ], and X-linked chronic granulomatous disease [ 35 ]. Unfortunately, many questions about the mechanisms of hiPS-derived macrophages in disease pathogenesis remain.…”

Induced Pluripotent Stem Cells as a Tool for Modeling Hematologic Disorders and as a Potential Source for Cell-Based Therapies

“…The researchers utilized recombinant NOX2/p22phox proteoliposomes containing an active cytochrome b558, the membrane component of NADPH oxidase complex, and delivered it to CGD-iPSC-derived macrophages. After treatment with specific liposomes, NADPH oxidase activity was restored without toxicity, making it a potential approach for future treatment of pulmonary infection in CGD patients [153]. For Fanconi anemia (FA), a congenital disorder characterized by bone marrow failure, FA-iPSCs were used for drug screening of several compounds known to improve FA phenotypes, such as resveratrol (Sirt1 activator), danazol (synthetic androgen), and doramapimod (p38 MAPK inhibitor), based on the evaluation of the effects on hematopoietic differentiation.…”