766. Genetic Correction of Ifnγr1 Deficiency in Hematopoietic Cells Repairs the Cellular Phenotype of Mendelian Susceptibility to Mycobacterial Disease

Mendelian Susceptibility to Mycobacterial Disease (MSMD) is a rare primary immunodeficiency characterized by recurrent severe infections with otherwise only weakly virulent mycobacteria. MSMD is associated with mutations in different genes, all leading to an impaired activation of macrophages by T-cells and a defective innate immune response. Specifically mutations in the interferon-gamma (IFNγ)-receptor-1 or-2 (IFNγR1 /2) genes result in a life-threatening disease phenotype with most patients dying in early childhood. We here introduce a novel gene therapy approach for IFNγR1 deficiency. Thus, we have designed a 3rd generation SIN lentiviral vector expressing the murine cDNA of Ifnγr1 from a spleen focus forming virus (SFFV) promoter element coupled by an IRES to GFP (Lv.SFFV.Ifnyr1.iGFP). Transduction of hematopoietic stem/progenitor cells from Ifnγrl−/− mice showed expression of Ifnγr1 by FACS and qRT-PCR and no abnormalities in clonogenic growth when compared to WT control cells. Moreover, differentiation of transduced cells towards macrophages (MΦ) by M-CSF was normal as determined by morphology on cytospins and surface marker expression of CD11b, CD200R, CD115, CD45 and F4/80. When subjecting corrected MΦ to stimulation with IFNγ, corrected cells were able to build a functional IFNγR1-Ifnγ-IFNγR2 and internalize IFNγ as efficiently as WT cells, as suggested by the clearance of IFNγ from the medium within 24h of stimulation. Consequently, Lv. SFFV. Ifnγr1.iGFP corrected MΦ revealed restored up-regulation of HLA-DR and CD86 (B7.2) comparable to WT-MΦ. IFNγ dependent T-cell activation was evaluated using T cell receptor- transgenic T cells recognizing ovalbumin (OVA). Both WT and corrected MΦ were unable to activate T-cells in the presence of IFNγ and OVA, whereas MΦ from Ifnγr1−/− mice induced significantly stronger proliferation of T cells. This observation was accompanied by the induction of indoleamine 2,3-dioxygenase (IDO) in WT and corrected MΦ, suggesting that IDO enables MΦ to deplete tryptophan from the medium and interferes with T-cell proliferation. Finally, Ifnγrl downstream signaling showed restored phosphorylation of STAT1 in corrected MΦ, consistent with induction of iNos and Irf1 upon stimulation with IFNγ. Moreover, correction of Ifnγrl−/− MΦ led to a significantly improved anti-mycobacterial activity as measured by comparable killing of Mycobacterium Avium in corrected and WT MΦ. Thus, we here introduce a novel gene therapy approach for MSMD in the context of Ifnγr1 deficiency.