Scavenger Receptor Class A1 Mediates Uptake of Morpholino Antisense Oligonucleotide into Dystrophic Skeletal Muscle

Figures

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  Figure 1. PMO Uptake into H2K-mdx52 Myotubes Is Mediated by Endocytosis H2K-WT and mdx52 myoblasts were differentiated for 4 days and treated with 5 or 10 μM PMO for 48 h (A and B) or 5 or 10 μM Cy5-conjugated PMO for 1, 4 (D and E), or 48 h (C). (A) RT-PCR analysis of exon 51 skipping with cDNA from the myotubes. A representative image is shown. M, 100-bp ladder marker. The dotted line shows the division of the gel. Exon-skipping efficiency was calculated using the densitometric value of the Δexon 51 plus 52 band divided by that of all bands (Δexon 51 plus 52 and Δexon 52) (n = 3 independent experiments). (B) H2K myotubes were preincubated with 1 mM NaN3 for 1 h and then supplemented with 5 μM PMO for 48 h in the presence of NaN3. The exon-skipping efficiency was calculated from the results of RT-PCR (n = 4 independent experiments). The unpaired t test was used for statistical analysis. (C) Representative image of RT-PCR after a 48-h treatment of H2K myotubes with 10 μM Cy5-PMO. The dotted line shows the division of the gel. (D) A representative image of Cy5 fluorescence in myotubes after 5 μM Cy5-PMO treatment for 1 or 4 h. The fluorescence intensities were quantified at each time point.…

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  Figure 2. Role of SR-A1 in PMO Uptake into H2K-mdx52 Myotubes (A) Gene expression of SR class A isoforms by qPCR using cDNA from H2K-WT and H2K-mdx52 myotubes 6 days after differentiation. The value of 2ΔCT is shown. Abt1 was used as a housekeeping gene. The Mann-Whitney U test was used for statistical analysis (n ≥ 3 independent experiments). (B) Western blotting of SR-A1 in the lysates of H2K-mdx52 myotubes on day 6 separated into cytosolic and membrane fractions. GAPDH was used as the control for the cytosolic fraction, and Na+/K+ ATPase and caveolin-3 were used as controls for the membrane fraction. Blots of each protein were derived from the same membrane. (C) Representative orthogonal sectioning of confocal images of caveolin-3 and SR-A1 in H2K-mdx52 myotubes on day 4 by the HyVolution method. Images of the xy, xz, and yz sections are shown. Scale bars, 10 μm. (D) H2K-mdx52 myotubes were pre-treated with SR inhibitors for 1 h and then supplemented with 5 μM PMO for 48 h in the presence of inhibitors. Ten micrograms per microliter poly I was used as an SR class A, C, E, and F inhibitor. Ten micrograms per microliter poly C was used as a control for poly I. Ten micrograms per…

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  Figure 3. Upregulated SR-A1 in Dystrophin-Deficient Mice Promotes PMO Uptake (A) Gene expression of SR class A isoforms in TA, GAS, and diaphragm (DP) muscles of 7- to 9-week-old WT and mdx52 mice as measured by qPCR. The Mann-Whitney U test was used for statistical analysis (n ≥ 3 independent experiments). (B) Immunohistochemistry of SR-A1 in TA and GAS muscles of 8- to 9-week-old WT, mdx52, and DKO mice. Scale bars, 200 μm. (C and D) Four-week-old WT, Mdx52, and DKO mice were intravenously injected with 75 mg/kg body weight of Cy5-PMO; 24 h later, TA, GAS, and diaphragm muscles were isolated. (C) Cy5 fluorescence in TA and GAS muscles was observed using the IVIS imaging system. Representative images are shown. Mean fluorescence intensity was quantified. One-way ANOVA was used for comparisons between multiple groups (n ≥ 6 independent experiments). (D) Cy5 fluorescence was observed in the sections of diaphragm muscles. Representative images of Cy5 fluorescence with DAPI are shown. Scale bars, 80 nm. (E) Four- to five-week-old mdx52 and DKO mice were intravenously injected with 160 mg/kg body weight of PMO; 2 weeks later, muscles were isolated, and their lysates were used for western…

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  Figure 4. Upregulated SR-A1 during Muscle Regeneration Increases PMO Localization in the Nucleus (A) Experimental model of intramuscular injection of Cy5-PMO after the induction of muscle regeneration with BaCl2. Mdx52 and DKO mice were intramuscularly injected with 1.2% BaCl2 in saline or only saline in the TA muscle of each leg. After 1 week, 50 μg of Cy5-PMO in saline was intramuscularly injected into both legs, and the muscles were collected 4 h later. (B) Gene expression of SR-A1 in TA muscles as measured by qPCR. Two-way ANOVA was used for comparisons between multiple groups (n ≥ 3 independent experiments). (C) Frozen sections were prepared for detection of Cy5-PMO under a fluorescence microscope. Representative images of Cy5 fluorescence merged with that of caveolin-3 and nuclear staining are shown. Scale bars, 80 μm.

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  Figure 5. PMOs with Negative Zeta Potential Bind to SR-A1 (A) The zeta potentials of 50 μM PMOs, the sequences of which are shown in Table 1, were measured in PBS, OptiMEM, and TAE at pH 7.4. One-way ANOVA was used for comparisons between multiple groups in each solvent (n ≥ 3 independent experiments). (B) Zeta potentials of all 10 PMOs in Table 1 measured in TAE at pH 7.4. The conductivity of each buffer is shown in Figure S9, and the sequence of each PMO is shown in Table 1. (C) The correlation between the zeta potential of PMOs in TAE buffer and the number of adenines subtracted from guanines plus thymines [(G + T) − A], cytosines plus adenines subtracted from thymines [T − (C+A)], and adenines subtracted from thymines (T − A). Pearson correlation coefficients and p values are indicated. (D) The zeta potentials of 50 μM PMOs, 51D, and poly A or poly T were measured in TAE at pH 7.4. The unpaired t test was used for statistical analysis (n ≥ 4 independent experiments). (E) PMOs (51D and 51A) were tested in a binding affinity assay with an N-terminal polyhistidine (His)-tagged recombinant SR-A1 by bio-layer interferometry. The anti-His tag biosensor was bound to SR-A1 and incubated with 1…