Design and synthesis of oligonucleotides
A series of 16mer ASOs were designed to target mouse Itga4 and Dmpk mRNA, Malat1 RNA, and human Stat3 mRNA. The ASOs had a 10 DNA nucleotide gapmer structure flanked by 3 LNA nucleotides. All internucleotide linkages were modified by phosphorothioate substitution to increase plasma ASO stability and protein binding, which ultimately increased tissue bioavailability68. Itga4-targeting ASO sequence: 5′-A^G^C^c^a^t^g^c^g^c^t^c^t^T^G^G-3′. Itga4-targeting scramble ASO sequence: 5′-G^C^G^a^c^g^c^g^t^t^g^a^c^T^C^T-3′ and 5′-G^C^G^g^c^g^a^t^c^t^a^t^g^C^T^C-3′. Dmpk mRNA, Malat1 RNA, and Stat3 mRNA targeting sequences: 5′-A^C^A^a^t^a^a^a^t^a^c^c^g^A^G^G-3′, 5′-C^T^A^g^t^t^c^a^c^t^g^a^a^T^G^C-3′, and 5’-C^T^A^t^t^t^g^g^a^t^g^t^c^A^G^C, respectively. Malat1-targeting scramble ASO sequence: 5′-A^C^G^t^g^a^t^c^g^c^c^t^t^A^T^A-3′. Malat1-targeting mismatch ASO sequence: 5′-C^T^G^g^t^g^c^a^g^t^g^a^a^T^G^C-3′. For all 16-mers, lowercase letters represent DNA, bold uppercase letters represent LNA (capital C denotes LNA 5-methylcytosine), and carets represent phosphorothioate linkages. For flow cytometry and confocal microscopy, Alexa Fluor 647 or Cy5 were covalently bound to the ASO 5′-ends.
A series of 16-mer cRNAs were designed to be complementary to the ASO sequences. Phosphorothioate-modified 2′-O-methyl sugar modifications were used in cRNA complementary to the ASO strand LNA for protection from exonucleases. To produce Toc-cRNA, α-tocopherol was covalently bound to the 5′-end of the cRNA strand (Fig. 1). To generate Toc-HDO, equimolar amounts of ASO and cRNA strands in PBS were heated at 95 °C for 5 min and slowly cooled to room temperature. All ASOs and cRNAs were synthesized by Gene Design (Osaka, Japan) or Hokkaido System Science (Sapporo, Japan).
Mouse studies
Animal experiments were performed at Tokyo Medical and Dental University. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (No. 0170179A). Experimental procedures were in accordance with the ethical and safety guidelines for animal experiments of Tokyo Medical and Dental University. Male wild-type C57BL/6 mice aged 6–9 weeks and BALB/c mice aged 10 weeks (Oriental Yeast, Tokyo, Japan) were kept on a 12-h light/dark cycle in a pathogen-free animal facility with free access to food and water (temperature: 18–24 °C; humidity: 40–70%). Toc-HDO, ASO, or PBS were administered by tail-vein injection according to body weight. For postmortem analyses, mice were deeply anesthetized with intraperitoneally administered pentobarbital (60 mg/kg) and sacrificed by transcardiac perfusion with PBS after confirming an absent blink reflex.
Induction of EAE
To induce active EAE, 9–10-week-old female C57BL/6J mice were immunized subcutaneously with 200 µg MOG35–55 peptide (MBL, Tokyo, Japan) and 400 µg Mycobacterium tuberculosis in incomplete Freund’s adjuvant (Difco Laboratories). Pertussis toxin (200 ng per mouse; List Biologicals) was injected intraperitoneally the same day and 2 days post-immunization. Animals were observed daily for clinical symptoms and scored by a masked investigator as follows: 0, no clinical disease; 1, tail weakness; 2, tail paralysis; 3, hindlimb weakness; 4, forelimb weakness; 5, forelimb paralysis; 6, moribund or death. To induce adoptive-transferred EAE, donor mice were immunized as described. The mice were euthanized 12 days after immunization, and the draining lymph nodes and spleen were harvested. Single-cell suspensions were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 50 µM 2-mercaptoethanol, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 µM MOG35-55 peptide, and 3 µM Toc-HDO or ASO targeting Itga4. After 72 h, cells were harvested and resuspended in Hanks’ balanced salt solution. A total of 1 × 107 viable cells in 100 µl were injected into the tail vein of wild-type mice. Mice also received two doses of pertussis toxin (200 ng per mouse) on the day of transfer and 2 days post-transfer.
MHC-mismatched allogeneic hematopoietic cell transplantation and clinical assessment of acute GVHD
MHC-mismatched allogeneic hematopoietic stem cell transplantation (B6 to BALB/c) was performed. Briefly, B6 mice (H-2b) were treated with PBS, Itga4-ASO, and Toc-HDO at corresponding doses of 50 mg/kg of ASO. Three days later, spleen T cells were isolated using a Pan T cell isolation Kit II for mouse (Miltenyi Biotec, Bergisch Gladbach, Germany). Bone marrow was flushed from non-treated B6 mice tibia and femur. TCD-BM were obtained using mouse CD3ε MicroBead Kit (Miltenyi Biotec) according to the manufacturer’s instructions. In all, 5 × 106 TCD-BM along with 1 × 106 spleen T cells were transplanted into lethally irradiated (900 cGy on day −1) allogeneic BALB/c recipient mice (H-2d). Recipients were monitored daily for survival and clinical GVHD score as described previously69. The scoring system for acute GVHD had six clinical criteria (maximum index = 11). Weight loss of <10% was scored 0, of >10% and <25% was scored as 1, and of >25% was scored as 2. For gastrointestinal symptoms, the scoring system denoted 0 as normal and 1 as suffering from diarrhea. For posture and activity, the scoring system denoted 0 as normal, 1 for hunching at rest and a mild to moderate decrease in activity, and 2 for severe hunching and a severe decrease in activity. For fur texture and skin integrity, the scoring system denoted 0 as normal, 1 for mild to moderate fur ruffling and scaling of the paws and tails, and 2 for severe fur ruffling and an obviously denuded skin. GVHD experiments were performed with four mice per group
In vivo studies
Mouse peripheral blood, splenic, and thymic lymphocytes were isolated using Lymphocyte Separation Medium 1077 (PromoCell, Heidelberg, Germany). Briefly, peripheral blood and cell suspensions were obtained by crushing the spleen and thymus. Suspensions were diluted with an equal volume of sterile PBS and the diluted cell suspension was carefully overlaid on three volumes of Lymphocyte Separation Medium 1077 in 15 ml tubes and centrifuged at 400 × g for 40 min without brakes. Mouse lymphocytes were removed from the liquid/medium interface and washed three times with 0.1% bovine serum albumin (BSA) in PBS.
Cell culture
The EL4 mouse T cell line and the Jurkat T cell line were obtained from the American Type Culture Collection and cultured as previously described70,71. Briefly, cells were cultured in RPMI 1640 containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-mercaptoethanol (all from Nacalai Tesque, Kyoto, Japan) in a humidified chamber at 37 °C containing 5% CO2.
Mouse peripheral blood and lymph node T cells were expanded from the cell suspension described above. Cells were re-suspended in RPMI-1640 containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-mercaptoethanol and incubated for 2 h in a humidified chamber at 37 °C containing 5% CO2. Non-adherent cells were collected and incubated for another 2 h to remove monocytes. Finally, non-adherent cells were washed three times and cultured with 1 µg/ml anti-CD3 antibody (BD Pharmingen, clone 145-2C11, #553058) and 2 µg/ml anti-CD28 antibody (BD Pharmingen, clone 37.51, #557393) for 2–3 days.
Treatment of cells with endocytosis inhibitors
Inhibitors were used at the following concentrations: amiloride: 1–3 mM; chlorpromazine: 5–30 µM; dynasore: 10–100 µM; filipin III, 1–3 µM; and cytochalasin D, 10–20 nM (all from Sigma-Aldrich). Cells were pretreated with each inhibitor for 1 h at 37 °C and then treated with 500 nM ASO or Toc-HDO for 4 h at 37 °C in the presence of inhibitors. Cells were then washed and cultured in fresh media without inhibitors for 20 h prior to RNA isolation. In experiments where ASO or Toc-HDO treatment was performed at 4 °C, the cells were incubated at specified temperatures for 1 h and then treated with ASOs or Toc-HDO for 4 h at the same temperature. Cells were washed, replenished with fresh media, and incubated for another 20 h at 37 °C prior to RNA isolation.
Stimulation of cytokine production
Primary T cells isolated as above were seeded at 1 × 106 cells in a 24-well plate and incubated with 1 µM Toc-HDO or ASO targeting Itga4 or Malat1, followed by supplementation with 50 ng/ml of phorbol myristate acetate and 500 ng/ml of ionomycin for 4 h at 37 °C with 5% CO2. After incubation, the cell suspensions were centrifuged and supernatants were analyzed for the presence of interferon-γ and TNF-α using an enzyme-linked immunosorbent assay using paired antibodies (eBioscience).
RNA isolation and quantitative real-time polymerase chain reaction (RT-PCR)
Total RNA was extracted from single-cell suspensions following incubation with gene-specific ASO or Toc-HDO using ISOGEN (Nippon Gene, Tokyo, Japan). To detect mRNA, RNA (1 µg) was reverse transcribed with Transcriptor Universal cDNA Master Mix (Roche Diagnostics). To estimate mRNA expression, quantitative RT-PCR analysis was performed using the TaqMan MicroRNA Reverse Transcription Kit, a Light Cycler 480 Real-Time PCR Instrument, and Light cycler 480 software (Roche Diagnostics). The primers and probes for mouse Dmpk (NM_032418.2), Epn2 (NM_001252188.2), Gfap (NM_001131020.1), Glyceraldehyde-3-phosphate dehydrogenase (Gapdh, NM_001289726.1), Iba1 (also known as Aif1, NM_001361501.1), Itga4 (NM_010576.4), Interferon-β (Ifn-β, NM_010510.1), Interferon-γ (Ifn-γ, NM_008337.4), Il-1b (NM_008361.4), Malat1 (NR_002847.3), and Tnf-α (NM_013693.3) were purchased from Thermo Fisher Scientific. Relative target gene mRNA levels were normalized to Gapdh mRNA levels.
Flow cytometry and fluorescence-activated cell sorting
The delivery efficiency of ASO or Toc-HDO into primary T cells in vivo and EL4 in vitro cells was assayed by flow cytometry using Alexa Flour 647-labeled ASO. Cells were harvested at the indicated time points and washed in PBS by centrifugation at 500 ×;g for 5 min. The cells were then washed and suspended in PBS with 2% BSA and 0.05% sodium azide (Sigma-Aldrich). Surface marker expression levels were determined using the following antibodies: CD3-PE (BioLegend, clone 17A2, 100205), CD45R/B220-PECy7 (BioLegend, clone RA3-6B2, #103221) or CD45R/B220-FITC (BioLegend, clone RA3-6B2, #103205), and CD49d-APC (BioLegend, clone R1-2, #103621). All related antibodies are listed in Supplementary Table 2. Cells were then sorted using a BD FACSAria cell sorter, BD FACSDiva software, and FlowJo v10 software (BD Biosciences) or analyzed using a BD FACSVerse cell analyzer and BD FACSuite software (BD Biosciences). The sorting/gating strategy is shown in Supplementary Fig. 2g.
Western blot analysis
Whole-cell extracts were isolated using RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)) supplemented with protease inhibitors (Roche). The proteins were separated on a 15% SDS–polyacrylamide gel electrophoresis gel and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore). Membranes were then incubated with the blocking buffer followed by incubation with the primary antibodies against Integrin α4 (Cell Signaling, clone D2E1, #8440), STAT3 (Cell Signaling, clone 124H6, #9139) or peroxidase-conjugated GAPDH (Wako, clone 5A12, #015-25473) and the appropriate secondary antibodies. Protein band intensities were quantified with the Fiji image processing software (National Institutes of Health)72.
Oligonucleotide concentration in lymphocytes
Following isolation of peripheral blood lymphocytes from mice injected with Alexa Fluor 647-conjugated ASO or Toc-HDO, detection assays were performed on a microplate reader Infinite M1000 PRO (Tecan, Männedorf, Switzerland). Oligonucleotide concentration in a cell was determined by comparing the measured fluorescence intensity with that of the Alexa Fluor 647-labeled reference oligonucleotides of known concentration and corrected with division by total cell counts.
Histopathological analyses of spinal cord tissues
For pathological analyses, all tissues were fixed in 10% neutral-buffered formalin solution for 24 h, embedded in paraffin, and cut into 5-µm sections. IHC staining was performed with anti-CD4 polyclonal antibody (NOVUS Biologicals, #NBP1-19371) and detected using Histofine Simple Stain Mouse MAX PO (R) (Nichirei Biosciences, Tokyo, Japan). For immunofluorescence (IF), slides were stained with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories) to visualize the nuclei and were immunolabeled with antibodies against myelin basic protein (Abcam, # ab40390) and Iba1 (Wako, #019-19741). Samples were then incubated with Alexa Fluor 488-conjugated (Invitrogen, #A11006) or Alexa Fluor 647-conjugated (Invitrogen, #A21244) secondary antibody. All IHC or IF antibodies are listed in Supplementary Table 2. For studies using Cy5- or Alexa Fluor 647-conjugated ASO and Toc-HDO, mouse lymphocytes were centrifuged directly onto glass slides and stained with DAPI. All images were acquired with an A1R confocal laser scanning microscope (Nikon, Tokyo, Japan) and BZ-X700 fluorescence microscope (Keyence, Osaka, Japan). Quantitative image analysis was performed using the Fiji image processing software (National Institutes of Health).
Statistical analysis
All data represent mean ± s.e.m. Differences among three groups were analyzed by one-way analysis of variance followed by Holm’s post hoc test. Statistical differences between two groups were analyzed using two-sided Student’s two-tailed t test. Dose response data were fitted to a four-parameter log-logistic curve using GraphPad Prism 8.3.0 and Fiji image processing software.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
