Interferon-α receptor antisense oligonucleotides reduce neuroinflammation and neuropathology in a mouse model of cerebral interferonopathy

Abstract

Chronic and elevated levels of the antiviral cytokine IFN-α in the brain are neurotoxic. This is best observed in patients with genetic cerebral interferonopathies such as Aicardi-Goutières syndrome. Cerebral interferonopathies typically manifest in early childhood and lead to debilitating disease and premature death. There is no cure for these diseases with existing treatments largely aimed at managing symptoms. Thus, an effective therapeutic strategy is urgently needed. Here, we investigated the effect of antisense oligonucleotides targeting the murine IFN-α receptor (Ifnar1 ASOs) in a transgenic mouse model of cerebral interferonopathy. Intracerebroventricular injection of Ifnar1 ASOs into transgenic mice with brain-targeted chronic IFN-α production resulted in a blunted cerebral interferon signature, reduced neuroinflammation, restoration of blood-brain barrier integrity, absence of tissue destruction, and lessened neuronal damage. Remarkably, Ifnar1 ASO treatment was also effective when given after the onset of neuropathological changes, as it reversed such disease-related features. We conclude that ASOs targeting the IFN-α receptor halt and reverse progression of IFN-α-mediated neuroinflammation and neurotoxicity, opening what we believe to be a new and promising approach for the treatment of patients with cerebral interferonopathies.

Keywords: Drug therapy; Inflammation; Innate immunity; Neurological disorders; Neuroscience.

Figure 1. ASO-mediated knockdown of Ifnar1 reduces neuropathology in GIFN mice.

(A) Schematic of experiment. (B) Real-time qPCR of relative Ifnar1 expression in cortex and spinal cord of mice. n = 7–8 mice per genotype per treatment. Mean and SEM shown. (C) Representative images of GFAP, Iba1, and CD3 IHC in the cortex and alizarin red S (ARS) and H&E staining in the cerebellum of vehicle and treated WT and GIFN mice. Scale bars: 100 μm and 20 μm in inserts. Arrowheads indicate CD3⁺ cells. (D) Quantification of the number of CD3⁺ T cells in brain sections normalized to area (n = 6–10 mice per genotype per treatment). (E) Relative level of serum neurofilament heavy subunit (NF-H) in treated WT and GIFN mice (n = 6–10 mice per genotype per treatment). Each point is a mouse. Mean and SEM shown. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-way ANOVA with Tukey’s post test.

Figure 2. Transcriptomic changes with ASO treatment in GIFN mice reflect improved neuropathology.

(A) Box plots of normalized expression levels (weighted trimmed mean of M values, TMM) of IFN-stimulated genes. Points are individual mice. ^, compared with corresponding treatment in WT mice; *, compared with corresponding ASO_c treatment, indicating P < 0.05 by quasi-likelihood F-test. (B) IFN score (n = 6–9 mice per genotype per treatment; each point is a mouse and mean and SEM are shown. ****P < 0.0001, by 2-way ANOVA with Tukey’s post test). (C) Mean-difference plots with key genes indicated that are associated with pathways in D. Gray dots represent genes that are not significantly regulated. (D) Predicted activation status of significantly enriched pathways identified by Ingenuity Pathway Analysis of all significantly regulated genes for each comparison.

Figure 3. Delayed Ifnar1 ASO dosing reduces Ifnar1 transcript and NF-H disease biomarker levels in GIFN mice with established neuropathology.

(A) Schematic of the experiment. Tail blood was taken 1 week prior to the second dose and/or 1 week prior to euthanasia. (B) Real-time qPCR of relative Ifnar1 expression in cortex and spinal cord of mice (n = 6–8 per genotype per treatment). Each point is a mouse and mean and SEM shown. 2-way ANOVA with Tukey’s post test. (C) Relative level of serum NF-H in treated WT and GIFN mice (n = 5–6 mice per genotype per treatment). Each point is a mouse and mean and SEM are shown. **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 1-way ANOVA with Tukey’s post test.

Figure 4. Delayed dosing of GIFN mice with Ifnar1 ASO reverses neuropathology.

(A) Quantification of CD3⁺ T cells in brain sections of 21-week-old (5 weeks after second dose) WT or GIFN mice treated with vehicle + vehicle, ASO_c + ASO_c, Ifnar1 ASO1 + Ifnar1 ASO1, or vehicle + Ifnar1 ASO1. Counts were normalized to area. Representative images of (B) GFAP and (C) Iba1 IHC in treated WT and GIFN mice with (D) quantification of microglia per mm² and (E) fibrinogen IHC to indicate blood-brain barrier leakage. Scale bar: 1 mm and 100 μm for high magnification. (A and D) Each point is a mouse and mean and SEM shown. *P < 0.05, **P < 0.01, and ****P < 0.0001, by 1-way ANOVA with Tukey’s post test.