ISG15 deficiency features a complex cellular phenotype that responds to treatment with itaconate and derivatives.

BACKGROUND: Congenital ISG15 deficiency is a rare autoinflammatory disorder that is driven by chronically elevated systemic interferon levels and predominantly affects central nervous system and skin. METHODS AND RESULTS: We have developed induced pluripotent stem cell-derived macrophages and endothelial cells as a model to study the cellular phenotype of ISG15 deficiency and identify novel treatments. ISG15-/- macrophages exhibited the expected hyperinflammatory responses, but normal phagocytic function. In addition, they displayed a multifaceted pathological phenotype featuring increased apoptosis/pyroptosis, oxidative stress, glycolysis, and acylcarnitine levels, but decreased glutamine uptake, BCAT1 expression, branched chain amino acid catabolism, oxidative phosphorylation, ß-oxidation, and NAD(P)H-dependent oxidoreductase activity. Furthermore, expression of genes involved in mitochondrial biogenesis and respiratory chain complexes II-V was diminished in ISG15-/- cells. Defective mitochondrial respiration was restored by transduction with wild-type ISG15, but only partially by a conjugation-deficient variant, suggesting that some ISG15 functions in mitochondrial respiration require ISGylation to cellular targets. Treatment with itaconate, dimethyl-itaconate, 4-octyl-itaconate, and the JAK1/2 inhibitor ruxolitinib ameliorated increased inflammation, propensity for cell death, and oxidative stress. Furthermore, the treatments greatly improved mitochondria-related gene expression, BCAT1 levels, redox balance, and intracellular and extracellular ATP levels. However, efficacy differed among the compounds according to read-out and cell type, suggesting that their effects on cellular targets are not identical. Indeed, only itaconates increased expression of anti-oxidant genes NFE2L2, HMOX1, and GPX7, and dimethyl-itaconate improved redox balance the most. Even though itaconate treatments normalized the elevated expression of interferon-stimulated genes, ISG15-/- macrophages maintained their reduced susceptibility to influenza virus infection. CONCLUSIONS: These findings expand the cellular phenotype of human ISG15 deficiency and reveal the importance of ISG15 for regulating oxidative stress, branched chain amino acid metabolism, and mitochondrial function in humans. The results validate ruxolitinib as treatment for ISG15 deficiency and suggest itaconate-based medications as additional therapeutics for this rare disorder.

Human Lentiviral Gene Therapy Restores the Cellular Phenotype of Autosomal Recessive Complete IFN-γR1 Deficiency.

Autosomal recessive (AR) complete interferon-γ receptor 1 (IFN-γR1) deficiency, also known as one genetic etiology of Mendelian susceptibility to mycobacterial disease (MSMD), is a life-threatening congenital disease leading to premature death. Affected patients present a pathognomonic predisposition to recurrent and severe infections with environmental mycobacteria or the Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine. Current therapeutic options are limited to antibiotic treatment and hematopoietic stem cell transplantation, however with poor outcome. Given the clinical success of gene therapy, we introduce the first lentiviral-based gene therapy approach to restore expression and function of the human IFN-γR-downstream signaling cascade. In our study, we developed lentiviral vectors constitutively expressing the human IFN-γR1 and demonstrate stable transgene expression without interference with cell viability and proliferation in transduced human hematopoietic cells. Using an IFN-γR1-deficient HeLa cell model, we show stable receptor reconstitution and restored IFN-γR1 signaling without adverse effect on cell functionality. Transduction of both SV40-immortalized and primary fibroblasts derived from IFN-γR1-deficient MSMD patients was able to recover IFN-γR1 expression and restore type II IFN signaling upon stimulation with IFN-γ. In summary, we highlight lentiviral vectors to correct the IFN-γ mediated immunity and present the first gene therapy approach for patients suffering from AR complete IFN-γR1 deficiency.

Effective hematopoietic stem cell-based gene therapy in a murine model of hereditary pulmonary alveolar proteinosis.

Hereditary pulmonary alveolar proteinosis due to GM-CSF receptor deficiency (herPAP) constitutes a life-threatening lung disease characterized by alveolar deposition of surfactant protein secondary to defective alveolar macrophage function. As current therapeutic options are primarily symptomatic, we have explored the potential of hematopoietic stem cell-based gene therapy. Using Csf2rb-/- mice, a model closely reflecting the human herPAP disease phenotype, we here demonstrate robust pulmonary engraftment of an alveolar macrophage population following intravenous transplantation of lentivirally corrected hematopoietic stem and progenitor cells. Engraftment was associated with marked improvement of critical herPAP disease parameters, including bronchoalveolar fluid protein, cholesterol and cytokine levels, pulmonary density on computed tomography scans, pulmonary deposition of Periodic Acid-Schiff+ material as well as respiratory mechanics. These effects were stable for at least nine months. With respect to engraftment and alveolar macrophage differentiation kinetics, we demonstrate the rapid development of CD11c+/SiglecF+ cells in the lungs from a CD11c-/SiglecF+ progenitor population within four weeks after transplantation. Based on these data, we suggest hematopoietic stem cell-based gene therapy as an effective and cause-directed treatment approach for herPAP.

Hematopoietic stem cell gene therapy for IFNγR1 deficiency protects mice from mycobacterial infections.

Mendelian susceptibility to mycobacterial disease is a rare primary immunodeficiency characterized by severe infections caused by weakly virulent mycobacteria. Biallelic null mutations in genes encoding interferon gamma receptor 1 or 2 (IFNGR1 or IFNGR2) result in a life-threatening disease phenotype in early childhood. Recombinant interferon γ (IFN-γ) therapy is inefficient, and hematopoietic stem cell transplantation has a poor prognosis. Thus, we developed a hematopoietic stem cell (HSC) gene therapy approach using lentiviral vectors that express Ifnγr1 either constitutively or myeloid specifically. Transduction of mouse Ifnγr1-/- HSCs led to stable IFNγR1 expression on macrophages, which rescued their cellular responses to IFN-γ. As a consequence, genetically corrected HSC-derived macrophages were able to suppress T-cell activation and showed restored antimycobacterial activity against Mycobacterium avium and Mycobacterium bovis Bacille Calmette-Guérin (BCG) in vitro. Transplantation of genetically corrected HSCs into Ifnγr1-/- mice before BCG infection prevented manifestations of severe BCG disease and maintained lung and spleen organ integrity, which was accompanied by a reduced mycobacterial burden in lung and spleen and a prolonged overall survival in animals that received a transplant. In summary, we demonstrate an HSC-based gene therapy approach for IFNγR1 deficiency, which protects mice from severe mycobacterial infections, thereby laying the foundation for a new therapeutic intervention in corresponding human patients.