Fig 2: Adoptive transfer of primary T cells treated with Toc-HDO ex vivo suppresses experimental autoimmune encephalomyelitis.a Quantitative RT-PCR analyses of Itga4 mRNA levels in MOG35-55 peptide-primed T cells incubated with 1 µM Toc-HDO, ASO, or PBS alone for 72 h without any transfection reagents (n = 3 for each group). b Clinical EAE scores of mice after transfer of MOG35-55-primed T cells treated with Toc-HDO, ASO or PBS alone. c Immunohistochemical staining for myelin basic protein (MBP) and Iba1 in mouse spinal cord sections after treatment with Toc-HDO, ASO, or PBS alone and wild-type (WT) mice. Scale bars, 100 µm. Images shown are representative of two experiments. d Percentage of MBP-positive area in the spinal cord white matter, and percentage of Iba1-postive area in the spinal cord. Data from WT (n = 3), PBS (n = 7), ASO (n = 4), and Toc-HDO (n = 5) for MBP-positive area and WT (n = 6), PBS (n = 6), ASO (n = 4), and Toc-HDO (n = 4) for Iba1-positive area e Quantitative RT-PCR analysis of Gfap, Iba1, and Il-1ß mRNA in lumbar spinal cord tissue of EAE mice after adoptive transfer of MOG35-55-primed T cells treated with Toc-HDO, ASO, or PBS alone (n = 4 for each group). Quantitative RT-PCR data are normalized to Gapdh mRNA levels. Data are expressed as mean ± s.e.m. Data represent at least two independent experiments. P values were calculated using one-way ANOVA with Holm’s post-test. Source data are provided as a Source data file.

Fig 3: Toc-HDO targeting Itga4 ameliorates clinical EAE symptoms and suppresses spinal cord demyelination and immune cell infiltration.a Clinical EAE scores of mice after prophylactic treatment with Toc-HDO, ASO, or PBS alone twice weekly for a total of five injections after immunization. b Immunohistochemical staining of CD4+ T cells in mouse spinal cord sections after treatment with Toc-HDO, ASO, or PBS alone. Scale bars, 50 µm. Images shown are representative of two experiments. c Immunohistochemical staining for myelin basic protein (MBP) and Iba1 in mouse spinal cord sections after treatment with Toc-HDO, ASO, or PBS alone, and wild-type (WT) mice. Scale bars, 100 µm. Images shown are representative of two experiments. d Percentage of MBP-positive area in spinal cord white matter, and the percentage of Iba1-postive area in spinal cord (n = 3 for each group). e Quantitative RT-PCR analysis of Gfap, Iba1, and Il-1ß mRNA in lumbar spinal cord tissue of EAE mice after treatment with Toc-HDO, ASO, or PBS alone, and WT mice (n = 4 for each group). f Clinical EAE scores of mice therapeutically treated with Toc-HDO, ASO, or PBS alone after EAE symptoms onset. g The severity of EAE disease evaluated by the area under the curve (AUC). Data from PBS (n = 9), ASO (n = 8), and Toc-HDO (n = 6). Data are expressed as mean ± s.e.m. and represent at least two independent experiments. P values were calculated using one-way ANOVA with Holm’s post-test. Source data are provided as a Source data file.

Fig 4: Gene silencing by intravenous administration of Toc-HDO targeting endogenous genes in mouse lymphocytes in vivo.a, b Target mRNA levels measured by quantitative RT-PCR in mouse lymphocytes from the indicated tissues 72 h after intravenous injection of 50 mg/kg Toc-HDO, ASO, or PBS alone. a Itga4. b Malat1. c Quantitative RT-PCR analyses of Malat1 RNA in mouse T cells and B cells from the indicated tissues 72 h after intravenous injection of 50 mg/kg Toc-HDO, ASO, or PBS alone (n = 4 for each group). d, e ITGA4 protein expression determined by flow cytometry in peripheral blood lymphocytes (d; n = 3 for each group) and by western blot in splenic lymphocytes (e; PBS, n = 4; ASO, n = 5; Toc-HDO, n = 6) 5 days after intravenous administration of 50 mg/kg Toc-HDO, ASO, or PBS alone. Quantitative RT-PCR data were normalized to Gapdh mRNA levels. Data are expressed as mean ± s.e.m. and represent at least two independent experiments. P values were calculated using one-way ANOVA with Holm’s post-test. Source data are provided as a Source data file.