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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 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.

SUBMITTER: Brault J

PROVIDER: S-EPMC5367562 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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

Brault Julie J Vaganay Guillaume G Le Roy Aline A Lenormand Jean-Luc JL Cortes Sandra S Stasia Marie José MJ

International journal of nanomedicine 20170320

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 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 i ...[more]

PMID: 28356734

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Project description:Myxobolus cerebralis is a cnidarian-myxozoan parasite that causes salmonid whirling disease. M. cerebralis alternates between two hosts: (1) a vertebrate salmonid and (2) an invertebrate oligochaete, Tubifex tubifex. There is no successful treatment for salmonid whirling disease. MyxSP-1 is a M. cerebralis serine protease implicated in whirling disease pathogenesis. We hypothesized that short-interfering RNA (siRNA)-induced RNA interference (RNAi) can silence MyxSP-1 in the invertebrate host and abrogate the M. cerebralis life cycle. This would preclude whirling disease infection in the salmonid host. To test this hypothesis, we first developed a siRNA delivery protocol in T. tubifex. Second, we determined the effective dose for siRNA treatment of M. cerebralis-infected T. tubifex. M. cerebralis-infected T. tubifex were treated with different concentrations of MyxSP-1 or negative control siRNAs (1μM, 2μM, 5μM or 7μM) at 15°C for 24h, 48h, 72h and 96h, respectively. We monitored MyxSP-1 knockdown using real-time quantitative PCR (qPCR). siRNA treatment with MyxSP-1 siRNA at 2μM concentration for 24h at 15°C showed maximum significant MyxSP-1 knockdown in T. tubifex. Third, we determined the time points in the M. cerebralis life cycle in T. tubifex at which siRNA treatment was most effective. M. cerebralis-infected T. tubifex were treated with MyxSP-1 or negative control siRNAs (2μM concentration for 24h at 15°C) at 24 hours post-infection (24hpi), 48hpi, 72hpi, 96hpi, 1 month post-infection (1mpi), 2mpi and 3mpi, respectively. We observed that siRNA treatment of T. tubifex was most effective at 1mpi, 2mpi and 3mpi. Fourth, we immersed specific-pathogen-free rainbow trout fry in water inhabited by MyxSP-1 siRNA-treated T. tubifex (at 1mpi, 2mpi and 3mpi). The salmonids did not develop whirling disease and showed significant MyxSP-1 knockdown. We also observed long-term RNAi in T. tubifex. Together these results demonstrate a novel RNAi-based therapeutic proof of concept in vivo against salmonid whirling disease.

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Dataset Information

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

Publications

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

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