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, 42 (5), 3207-17

Cellular Trafficking Determines the Exon Skipping Activity of Pip6a-PMO in Mdx Skeletal and Cardiac Muscle Cells

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Cellular Trafficking Determines the Exon Skipping Activity of Pip6a-PMO in Mdx Skeletal and Cardiac Muscle Cells

Taavi Lehto et al. Nucleic Acids Res.

Abstract

Cell-penetrating peptide-mediated delivery of phosphorodiamidate morpholino oligomers (PMOs) has shown great promise for exon-skipping therapy of Duchenne Muscular Dystrophy (DMD). Pip6a-PMO, a recently developed conjugate, is particularly efficient in a murine DMD model, although mechanisms responsible for its increased biological activity have not been studied. Here, we evaluate the cellular trafficking and the biological activity of Pip6a-PMO in skeletal muscle cells and primary cardiomyocytes. Our results indicate that Pip6a-PMO is taken up in the skeletal muscle cells by an energy- and caveolae-mediated endocytosis. Interestingly, its cellular distribution is different in undifferentiated and differentiated skeletal muscle cells (vesicular versus nuclear). Likewise, Pip6a-PMO mainly accumulates in cytoplasmic vesicles in primary cardiomyocytes, in which clathrin-mediated endocytosis seems to be the pre-dominant uptake pathway. These differences in cellular trafficking correspond well with the exon-skipping data, with higher activity in myotubes than in myoblasts or cardiomyocytes. These differences in cellular trafficking thus provide a possible mechanistic explanation for the variations in exon-skipping activity and restoration of dystrophin protein in heart muscle compared with skeletal muscle tissues in DMD models. Overall, Pip6a-PMO appears as the most efficient conjugate to date (low nanomolar EC50), even if limitations remain from endosomal escape.

Figures

Figure 1.
Figure 1.
Exon-skipping efficiency of Pip6a-PMO in mdx skeletal muscle cells. H2k mdx cells were differentiated for 5 days and thereafter incubated with Pip6a-PMO under serum-free (–S) (A) or serum conditions (+S) (C). Graphical view and calculation of half-maximal effective concentration (EC50) at 24 h post-treatment for exon skipping under serum-free and serum condition (B). EC50 values were calculated using Prism 5.0 software after normalization of the Δ23 skipping. Pip6a-PMO-meditated exon skipping in H2k mdx myoblasts incubated under serum conditions (+S) (D). Transfections were carried out for 4 h under serum-free or serum condition and cells were incubated further with serum-containing medium for 20 h. Exon-skipping efficiency was evaluated on pre-mRNA levels by nested RT-PCR. Products were separated by gel electrophoresis and exon-skipping values were derived from densitometric analysis. For each experiment, percentage of exon skipping (%ES) is calculated on the densitometric value of the Δ23 band related to the other bands (full length + Δ23 + Δ23 + 22) with n ≥ 4.
Figure 2.
Figure 2.
Comparative analysis of Pip6a-PMO uptake and exon skipping in H2k mdx skeletal muscle cells. Differentiated and non-differentiated H2k mdx cells were incubated 4 h with Pip6a-PMO at the indicated concentrations. The dose-depended internalization was assessed by fluorescence spectroscopy (A). Using the same differentiated H2K mdx samples, exon-skipping efficiency cells was evaluated by RT-PCR (B). (%ES calculated as described in Figure 1 with n ≥ 4).
Figure 3.
Figure 3.
Intracellular distribution of Pip6a-PMO in H2k mdx myoblasts and myotubes. Representative images of H2K mdx myoblasts or myotubes which were incubated with fluorescein-labelled Pip6a-PMO (1000 nM) or PMO (1000 nM) for the indicated times. Cell nuclei were labelled with Hoechst dye. Untreated cells are shown as controls. Pip6a-PMO or naked PMO distribution in live unfixed cells was evaluated by fluorescence microscopy with a Zeiss Axiovert 200 M. White bar = 10 µm.
Figure 4.
Figure 4.
Cellular trafficking of Pip6a-PMO in mdx skeletal muscle cells. Effect of energy depletion and endocytosis inhibitors on the uptake of Pip6a-PMO (at 250 nM) in H2k mdx myotubes at 4 h post-treatment as measured by fluorescence spectroscopy (n ≥ 4) (A) with the corresponding exon skipping activity of the same samples (B). (%ES calculated as described in Figure 1). Treatment with inhibitors was started 30 min before the treatment with Pip6a-PMO. The following inhibitors were used at indicated concentrations: NaN3 (10 mM) and 2′-Deoxy-D-Glucose (DDG) (6 mM) for ATP-depletion (-ATP); chlorpromazine (CPZ, 30 µM) for clathrin-mediated endocytosis inhibition; nystatin (Nys, 50 µM) for caveolae-mediated endocytosis inhibition; and 5-(N-ethyl-N-isopropyl) amiloride (EIPA, 10 µM) for macropinocytosis inhibition (n ≥ 4). (C) Effect of energy depletion and endocytosis inhibitors on the uptake of Pip6a-PMO (at 500 nM) in H2k mdx myoblasts at 4 h post-treatment as measured by fluorescence spectroscopy. For myoblasts chlorpromazine (CPZ) and nystatin (Nys) were used at 15 µM and 25 µM, respectively (n ≥ 4).
Figure 5.
Figure 5.
Cellular trafficking and exon-skipping efficiency of Pip6a-PMO in heart muscle cells. (A) Representative images of the cellular distribution of Pip6a-PMO in primary wild-type (WT) and mdx cardiomyocytes at 4 h as measured by fluorescence microscopy (Pip6a-PMO and PMO used at 1000 nM). Cell nuclei were labelled with Hoechst dye. Untreated cells are shown as controls. White bar = 10 µm. Exon-skipping efficiency of Pip6a-PMO in serum-containing medium at 24 h post-treatment in primary WT (B) and mdx (C) cardiomyocytes (n ≥ 4). RT-PCR analysis and %ES calculation as described in Figure 1.
Figure 6.
Figure 6.
Effect of endocytosis inhibitors on Pip6a-PMO uptake at 4 h post-treatment. Effect of energy depletion and endocytosis inhibitors on the uptake of Pip6a-PMO (at 1000 nM) in wild-type cardiomyocytes at 4 h post-treatment as measured by fluorescence spectroscopy. The following inhibitors were used at the indicated concentrations: NaN3 (10 mM) and 2′-Deoxy-D-Glucose (DDG) (6 mM) for ATP-depletion (-ATP); chlorpromazine (CPZ, 15 µM) for clathrin-mediated endocytosis inhibition; nystatin (Nys, 25 µM) for caveolae-mediated endocytosis inhibition; and 5-(N-ethyl-N-isopropyl) amilorid (EIPA, 10 µM) for macropinocytosis inhibition (n ≥ 4).

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