Background: Type I interferonopathies are a group of rare monogenic diseases causing over-activation of signaling pathways ultimately leading to increased type I Interferon (IFN) in the plasma and over-activation of the immune system. AGS is a rare inflammatory disease with onset at birth or early infancy that is principally caused by mutations in genes encoding proteins with cytosolic nuclease activity e.g., RNAseH2A, -B and -C and Trex1. Mutations in Trex1 lead to accumulation of DNA in the cytosol which activates the canonical DNA-sensing cGAS-STING pathway. SAVI, another rare and severe pediatric type 1 interferonopathy, is caused by mutations in the gene encoding the STING protein resulting in constitutive STING activation. Unlike AGS linked to Trex1, SAVI mutations drive a non-canonical activation of STING. In order to inhibit both canonical and non-canonical STING pathway activation, targeted protein degraders of STING have been designed and optimized as potential future treatments for AGS, SAVI and other diseases associated with excessive STING activation.

Objectives: The aims of this study were to characterize gene expression patterns in patient fibroblasts from AGS and SAVI patients, and to evaluate the therapeutic potential of a targeted STING protein degrader.

Methods: Gene expression in primary fibroblasts from AGS patients carrying RNaseH2B mutations (n=2) or a Trex1 mutation (n=1), SAVI patients carrying the N154S mutation (n=3), and healthy donors (HD) (n=3) were characterized by RNA sequencing and RT-qPCR. Gene expression from RT-qPCR was calculated using the 2 ^–(ΔΔCt) method. Selected genes were further assessed in cells treated with a targeted STING degrader, SP2C. Murine Trex1 KO bone marrow-derived macrophages (BMDM) treated with SP2C, were also analyzed by RT-qPCR for effects on gene expression.

Results: Expression of IFI27 was significantly upregulated (3-20 fold, p < 0.05) compared to HD in 3/3 SAVI patients and IFNB1 (2-4 fold) , ISG15 (1.5 fold) and CXCL10 (2-3 fold) in 2/3 SAVI patients (p < 0.05). In 3/3 AGS patient fibroblasts, gene expression of IFNB1 (1.5-2.5 fold) and CXCL10 (2.6 - 6 fold) were significantly upregulated compared to HD. Furthermore, IFIT1 (5 fold) , ISG15 (1.5-2.5 fold) , IFI27 (>100 fold) , IFI44 (7 fold) and IFI44L (5 fold) were all significantly upregulated compared to HD in AGS-Trex1 (p < 0.05) but not in AGS-RNAseH2B patients. Stimulation with a STING agonist boosted overall expression of interferon stimulated genes in both SAVI and AGS fibroblasts. The targeted protein degrader, SP2C, which degrades wild-type STING in human fibroblast (HFF-1) cells in a time-, concentration- and proteasome-dependent manner with a DC ₅₀ value of 210nM, was shown to degrade STING in both HD and SAVI fibroblasts. At a concentration of 1μM, it inhibited CXCL10, IFI27, ISG15, IFI44 and IFNB1 gene expression in both SAVI and AGS-Trex1 but not AGS-RNAseH2B patient fibroblasts. Findings in Trex1-mutant AGS were consistent with data from murine Trex1 KO BMDMs, where IFIT1 , CXCL10 and ISG15 gene expression was significantly increased relative to wildtype BMDM (p<0.0001) and could be significantly inhibited by SP2C (p<0.0001).

Conclusion: Expression of interferon-stimulated genes and genes encoding other pro-inflammatory cytokines is elevated in primary fibroblasts from AGS and SAVI patients. Expression of these genes in SAVI and Trex1-mutant AGS cells is inhibited by a targeted STING degrader, supporting the further development of this modality for the treatment of these diseases and other STING-mediated inflammatory diseases.

REFERENCES: NIL.

Acknowledgements: NIL.

Disclosure of Interests: Kristina Byskov Sulis Tx Aps, Pernille R. Noer Sulis Tx Aps, Emil H. Nilsson Sulis Tx Aps, Ulrich Sensfuss Sulis Tx Aps, Rune T. Kidmose Sulis Tx Aps, Alexander Haughuus Sulis Tx Aps, Helene K. Mortensen Sulis Tx Aps, Martin Roelsgaard Jakobsen Sulis Tx Aps, Sulis Tx ApS, Unikum Therapeutics ApS, Richard C. Bethell GSK plc, Sulis Tx Aps.