Antisense oligonucleotides
PMOs were synthesized by Gene Tools, LLC (Philomath, OR). All complementary strands for the experiment were synthesized by GeneDesign (Osaka, Japan). PMOs target the donor splice site of exon 23 (+7–18) of the mouse dystrophin pre-mRNA, 5’-GGCCAAACCTCGGCTTACCTGAAAT-3’.
Mouse studies
All animals were maintained on a 12 h light/12 h dark cycle in a pathogen-free animal facility (temperature: 18–24 °C; humidity: 40–70%) with free access to food (CLEA Rodent Diet CE-2, (CLEA Japan, Inc., Japan)) and water ad libitum. Mice (mdx, C57BL/10ScSn-Dmdmdx/J, 6–8 week-old males, and C57BL/10ScNJic [B10], 6–8 week-old males) were injected intravenously in the retro-orbital sinus or subcutaneously once per week with AONs, under general anesthesia using isoflurane. They were randomly assigned to experimental or control groups. All studies were conducted in accordance with the ethical guidelines of Tokyo Medical and Dental University, and in strict compliance with the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions as set forth by the Ministry of Education, Culture, Sports, Science and Technology. Approval for the experiments was granted by TMDU (Approval number A2022-085A). Experiments are in accordance with the ARRIVE guidelines. All possible efforts were made to minimize the number of animals used and to alleviate their discomfort.
Estimation of skipping efficiency in vivo
Each antisense oligonucleotide (AON) against exon 23 of the dystrophin gene was dissolved in PBS (stock concentrations: 2 mM), and 11.88 μmol/kg of each AON was injected into the retro-orbital sinus once weekly for a total of 1, 3, or 5 doses. Two weeks after the last injection, mice were sacrificed under anesthesia with 4% isoflurane (Wako, Osaka, Japan), and the muscles, brain, liver, and kidneys were dissected. Total RNA was extracted from cells or muscle tissues using ISOGEN 2 (NIPPON GENE, Tokyo, Japan), and 300 ng or 500 ng of total RNA was processed using the QIAGEN OneStep RT-PCR Kit (QIAGEN, Venlo, Nederland), according to the manufacturer’s instructions. The primer sequences were mEx22F 5’-ATCCAGCAGTCAGAAAGCAAA-3’ and mEx24R 5’-CAGCCATCCATTTCTGTAAGG-3’ for amplification from exons 22 to 24. The PCR conditions were 50 °C for 30 min and 95 °C for 15 min, 35 cycles of 94 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min, and finally, 72 °C for 7 min. The PCR bands were analyzed using Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA), and the resulting PCR bands were extracted using a QIAquick Gel extraction Kit (QIAGEN, Venlo, Nederland) for direct sequencing using an ABI 3100 (Thermo Fisher Scientific, Waltham, MA, USA) to confirm the exon skipping. Skipping efficiency was calculated using the following formula:
[(molality of skipped translation products) × 100% / (molality of skipped translation products + molality of unskipped translation products)].
Immunohistochemistry
Ten-micrometer cryosections were cut from flash-frozen muscle using the Leica CM3050 S, (Leica, Wetzlar, Germany) placed on MAS-coated glass slides (Matsunami Glass Industrial, Osaka, Japan), air-dried, and blocked for 1 h with 5% goat serum (S-1000 Vector Laboratories, Burlingame, CA, USA) in PBS or mouse-on-mouse blocking buffer containing mouse IgG blocking reagent (#MKB-2213 Vector Laboratories) at room temperature (~25 °C). The tissues were then incubated with the following primary antibodies overnight at 4 °C: rabbit anti-dystrophin against C-terminus (ab15277, 1:300; Abcam), mouse anti-ɑ-sarcoglycan (NCL-L-a-SARC, 1:200; Leica Biosystems), mouse anti-β-dystroglycan (NCL-b-DG, 1:200; Leica Biosystems), nNOS (#61-7000-rabbit, 1:1000; Thermo Fisher Scientific), and mouse anti-Caveolin 3 (sc-5310, 1:500; Santa Cruz Biotechnology). Subsequently, tissue sections were treated with secondary antibodies (Alexa Fluor 546 goat anti-mouse #A-11030 and Alexa Fluor 568 goat anti-rabbit #A-11011 Thermo Fisher Scientific) for 1 h (1:1000), protected from light at room temperature. Coverslips were mounted using VECTASHIELD Antifade Mounting Medium with 4’,6-diamidino-2-phenylindole (DAPI) (VECTOR H-1200). Centrally nucleated fibers and the myofiber cross-sectional area of QF were measured using HALO® Image Analysis (Indica labs, Albuquerque, NM, USA) (28).
In situ hybridization to the morpholino oligomer
In situ hybridization of the morpholino oligomer was performed using the miRNAscope® HD (RED) Assay Kit (Advanced Cell Diagnostics [ACD], Westminster, CO, USA), according to the manufacturer’s instructions. Fresh-frozen QF muscles and heart tissues were sectioned (10 μm) using the Leica CM3050 S, (Leica, Wetzlar, Germany) and placed on SuperFrost Plus slides (Thermo Fisher Scientific). Slides were fixed in 4% paraformaldehyde for 1 h at 4 °C, incubated in 50% ethanol for 5 min, 70% ethanol for 5 min, and washed in 100% ethanol twice for 5 min each. Sections were then incubated in hydrogen peroxide for 10 min at room temperature and washed in distilled water twice for 1 min each. Protease IV treatment was applied to the tissues, which were incubated in a chamber at room temperature for 30 min, followed by washing with PBS for 1 min. Slides were incubated with PMO sequence probes (SR-ASO-PMO-S1, #1088271-S1, ACD) for 2 h at 40 °C. Further amplification of the target probe signal was performed according to the manufacturer’s instructions (miRNAscope HD detection protocol Amp 1-6). Fast red was prepared by combining Red-A and Red-B (1:60), added to the sections, and incubated for 10 min at room temperature. Slides were mounted with EcoMount (EM897L, Biocare, Pacheco, CA, USA), counterstained with hematoxylin, and imaged on SLIDEVIEW VS200 (Evident Co., Tokyo, Japan) at ×40 magnification.
In situ hybridization chain reaction (HCR)
Tissue sections were prepared from formalin-fixed paraffin-embedded blocks. Deparaffinization of slides was performed in xylene and ethanol solutions at room temperature (25 °C). The sections were pretreated in Tris-EDTA Buffer, pH 9.0 (ab93684, abcam, Cambridge, UK) with microwave oven for 5 min and another 5 min for antigen retrieval. The sections were left immersed in the buffer at room temperature for at least 20 min and then slowly cooled. The slides were block with 3% bovine serum albumin (017-22231, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) in phosphate-buffered saline for 15 min. The mixture of anti-lamin A/C antibody (1:100, 8617 s, Cell Signal Technology, MA, US) and wheat germ agglutinin lectin (1:300, W11262, Thermo Fisher Scientific Inc., MA, US) was added to the slides and incubated overnight at 4 °C to stain nuclear membrane and cell boundary. In situ hybridization chain reaction was conducted to evaluate the distribution of PMO as Zhuang et al. with minor modifications30. Briefly, the hybridization of probe to PMO was performed according to the miRCURY® LNA® miRNA ISH Optimazation Kits (FFPE) protocol (Qiagen, Hilden, Germany). The LNA-modified probe with overhang for signal amplification was designed and synthesized at Qiagen. The sequence is 5-CTCTATATCTCCAACCCGAATTTCAGGTAAGCCGAGGTTT-3’. Slides were washed in 5X SSCT for 10 min and then incubated in amplification buffer (5X SSCT, 0.1 % Tween 20, 10% low molecular weight dextran sulfate) for 30 min at room temperature Hybridization chain reaction was performed in amplification buffer containing 6 μmol/L hairpin amplifiers. Slides were mounted in Prolong Diamond with DAPI (P36966, Thermo Fisher Scientific Inc.). Sections were imaged on an STELLARIS 8 confocal microscope (Leica Microsystems, Wetzlar, Germany). Imaging analysis was conducted with Imaris (ver. 9.7.0, Oxford Instruments, Abingdon, UK).
Western blotting
Proteins were extracted from sliced frozen muscle using SDS buffer (0.125 M Tris/HCl with pH 6.4, 10% glycerol, 4% SDS, 4 M urea, 10% ß-ME, and 0.005% BPB) supplemented with 1X Protease Inhibitor (Complete Mini, Roche Diagnostics, Mannheim, Germany). The normal control lysate from a B10 mouse was prepared as a reference for dystrophin expression. Subject and normal control lysates were denatured at 100 °C for 3 min and electrophoresed in a Tris-acetate 3–8% gradient polyacrylamide gel (Thermo Fisher Scientific) at 150 V for 40 min. The proteins were transferred to a PVDF membrane (Bio-Rad, Hercules, CA) via wet transfer at 30 V overnight. After incubation with 5% nonfat milk (NACALAI TESQUE, INC., Kyoto, Japan) in TBST for 60 min, the membrane was incubated at 4 °C overnight with an anti-dystrophin antibody (ab15277, 1:200; Abcam, Cambridge, UK) or anti-vinculin antibody (NB600-1293, 1:10000; Novus Biologicals, Centennial, CO, USA). The membrane was washed three times for 10 min each in TBST and incubated with a horseradish peroxidase-conjugated anti-rabbit (#111-035-003, 1:3000) or anti-mouse (#115-035-003, 1:10,000) antibodies (Jackson ImmunoResearch, West Grove, PA, USA) for 60 min, followed by six washes with TBST and allowed to develop with West Dura Extended Duration Substrate (Thermo Fisher Scientific), according to the manufacturer’s protocols. The immunoreactive bands were detected using the ChemiDoc XRS Image System (Bio-Rad Laboratories, Hercules, CA, USA).
Measurement of the PMO concentration
HELISA was performed based on the method previously reported by Burki et al.29 and Lim et al.61. PMOs in the blood were quantified using sera from blood samples of treated mdx mice or age-matched samples. For PMO uptake quantification, frozen muscle sections were weighed, homogenized in RIPA buffer (Thermo Fisher Scientific), and incubated with proteinase K (NACALAI TESQUE, INC., Kyoto, Japan) overnight at 55 °C. Then, lysates were spun at maximum speed for 15 min to collect the supernatant. Probes with complementary sequences to the PMOs used were synthesized and conjugated at the 5′ and 3′ ends with digoxigenin and biotin, respectively (GeneDesign, Osaka, Japan). The first and last seven nucleotides of the probes were fully phosphorothioated. PMO amounts were calculated in reference to a standard curve constructed from fluorescence values given by the respective PMO standards.
Estimation of fibrosis
Frozen tissue sample sections were stained with the Trichrome Stain Kit (Modified Masson’s: ScyTek Laboratories), according to the manufacturer’s instructions. Sections were stained with preheated Bouin’s Fluid for 60 min, cooled for 10 min, and washed twice with water. Then, slides were stained with working Weigert’s Iron Hematoxylin for 5 min, washed with water for 2 min, and Biebrich Scarlet/Acid Fuchsin Solution was applied to slides for 15 min. After washing with water, slides were differentiated in Phosphomolybdic/Phosphotungstic Acid Solution for 10–15 min, followed by Aniline Blue Solution for 5–10 min, and washed in water. Images were obtained by SLIDEVIEW VS200 (Olympus, Japan) at × 20 magnification. Blue areas of the transverse mdx mouse heart sections were measured at the papillary muscle level as areas of fibrosis; these values were then divided by the total cross-sectional area to determine the degree of cardiac fibrosis using HALO® Image Analysis (Indica labs, Albuquerque, NM)62.
Muscle function analysis
Forelimb grip test
Muscle strength was measured using the forelimb grip test with a grip strength meter (MK-380CM/FM; Muromachi Kikai, Co., Ltd., Tokyo, Japan). The average of three measurements per animal per time point was recorded for comparative analysis.
Treadmill exercise
Running sessions were performed on a four-lane motorized treadmill equipped with electric shock (Treadmill for Rats and Mice Model MK-680 S; Muromachi Kikai Co., Ltd) at least 1 week after the last injection. The treadmill was set at an inclination of 0°. All mice were acclimated to the treadmill belt for 5 min before starting to walk and then forced to run at 5 m/min for 5 min. Subsequently, the speed was increased by 1 m/min each minute. The test was stopped when the mouse was exhausted, did not attempt to remount the treadmill, or spent 5 continuous seconds on the shock grid, and the time to exhaustion was determined.
Restraint-induced unconditioned fear
All mice were tested between 10:00 am and 1:00 pm. Mice were restrained by the experimenter by placing the neck between the thumb and index finger and positioning the tail between the third and little fingers. After 10 s, the mouse was released into the observation box illuminated with 20 lux. Videos were captured by a camera placed on top of the box for 10 min using Image OF (O’Hara & Co. Ltd., Tokyo, Japan) and then transmitted to a personal computer (Panasonic, Japan). Total distance traveled and immobile time were measured. Complete immobilization of the mouse, except for respiration, was regarded as a freezing response63. This was quantified as the time the mouse moved <0.5 cm (2 cm) per second. Unconditioned fear responses induced by this acute stress were characterized by periods of tonic immobility (freezing) during the 10 min recording period.
Electrocardiography
Body-surface electrocardiography (ECG) was performed in a blinded manner, as described previously63. ECG in lead II configuration was recorded using the PowerLab system (PowerLab 4/26, ADInstruments) under anesthesia with 1% isoflurane. ECG parameters were obtained by averaging those from three different ECGs. The QT interval was defined as an interval between the onset of the QRS complex and the end of the negative component of the T wave. QTc was calculated using the following formula: QTc = QT interval (ms)/√(RR interval (s) × 10).
Blood chemistry and complete blood count analysis
Blood chemistry was assessed in the SRL Laboratory (Tokyo, Japan), and the blood cell count was measured at LSI Medicine (Tokyo, Japan).
Size and zeta-potential measurements
The size and size distribution of nanoparticles were determined via DLS using a Zetasizer Pro instrument (Malvern Instrument Ltd., UK) with an incident light (633 nm). The sample solutions were loaded into a low-volume cuvette (ZEN2112), and the measurements were carried out with a detection angle of 173° and a temperature of 25 °C.
Fluorescence polarization
Binding of PMOs and lipid-conjugate PMOs/HDOs to mouse albumin was determined using fluorescence polarization (FP), as described by Gaus et al.64. Briefly, PMO and PMO/HDO were labeled at the 5′ terminus of the PMO with Alexa Fluor 647. Binding measurements were conducted in 1X DPBS (Gibco) in flat-bottom non-binding 96-well plates (Corning, NY, USA) at 25 °C. Alexa 647-labeled PMO or PMO/HDO were added at a final concentration of 2 nM to solutions of albumin ranging from sub nM to mM concentrations. Solutions were equilibrated at least 30 min before measuring fluorescence polarization (λex = 635 nm, λem = 675 nm) on a Tecan InfiniteM1000 Pro (Baldwin Park, CA, USA).
Statistical analyses
The GraphPad Prism 9 software (version 9.5.0) and Microsoft Excel for Microsoft 365 MSO (version 2211) were used to analyze the data. All numerical values were presented as mean ± standard error of the mean (SEM). Differences among more than three groups were analyzed using one-way analysis of variance followed by Tukey’s Kramer tests. Statistical differences between two groups were analyzed using the Student’s one-tailed t-test. Significant levels were set at *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
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- Source: https://www.nature.com/articles/s41467-024-48204-5