Wu, B, Lu, P, Benrashid, E, Malik, S, Ashar, J, Doran, TJ et al.. Dose-dependent restoration of dystrophin expression in cardiac muscle of dystrophic mice by systemically delivered morpholino. Gene Ther 17: 132-140

McColl-Lockwood Laboratory for Muscular Dystrophy Laboratory, Neuromuscular/ALS Center, Department of Neurology, Carolinas Medical Center, Charlotte, NC 28231, USA.
Gene therapy (Impact Factor: 3.1). 09/2009; 17(1):132-40. DOI: 10.1038/gt.2009.120
Source: PubMed


We have earlier shown that antisense morpholino oligomers are able to restore dystrophin expression by systemic delivery in body-wide skeletal muscles of dystrophic mdx mice. However, the levels of dystrophin expression vary considerably and, more importantly, no dystrophin expression has been achieved in cardiac muscle. In this study, we investigate the efficiency of morpholino-induced exon skipping in cardiomyoblasts and myocytes in vitro, and in cardiac muscle in vivo by dose escalation. We showed that morpholino induces targeted exon skipping equally effectively in both skeletal muscle myoblasts and cardiomyoblasts. Effective exon skipping was achieved in cardiomyocytes in culture. In the mdx mice, morpholino rescues dystrophin expression dose dependently in both skeletal and cardiac muscles. Therapeutic levels of dystrophin were achieved in cardiac muscle albeit at higher doses than in skeletal muscles. Up to 50 and 30% normal levels of dystrophin were induced by single systemic delivery of 3 g kg(-1) of morpholino in skeletal and cardiac muscles, respectively. High doses of morpholino treatment reduced the serum levels of creatine kinase without clear toxicity. These findings suggest that effective rescue of dystrophin in cardiac muscles can be achieved by morpholino for the treatment of Duchenne muscular dystrophy.

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    • "As mentioned before, skeletal muscles become permeable in the absence of dystrophin, facilitating the uptake of AONs. The heart is built up of individual cardiomyocytes, which do not display this leakiness, thereby making it harder for AONs to get inside.12,28 The assay used for determination of 23AON levels does not discriminate between AON inside the heart as whole organ and AON that is actually inside the cardiomyocytes. "
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    ABSTRACT: Antisense-mediated exon skipping is currently in clinical development for Duchenne muscular dystrophy (DMD) to amend the consequences of the underlying genetic defect and restore dystrophin expression. Due to turnover of compound, transcript, and protein, chronic treatment with effector molecules (antisense oligonucleotides) will be required. To investigate the dynamics and persistence of antisense 2'-O-methyl phosphorothioate oligonucleotides, exon skipping, and dystrophin expression after dosing was concluded, mdx mice were treated subcutaneously for 8 weeks with 100 mg/kg oligonucleotides twice weekly. Thereafter, mice were sacrificed at different time points after the final injection (36 hours-24 weeks). Oligonucleotide half-life was longer in heart (~65 days) compared with that in skeletal muscle, liver, and kidney (~35 days). Exon skipping half-lives varied between 33 and 53 days, whereas dystrophin protein showed a long half-life (>100 days). Oligonucleotide and exon-skipping levels peaked in the first week and declined thereafter. By contrast, dystrophin expression peaked after 3-8 weeks and then slowly declined, remaining detectable after 24 weeks. Concordance between levels of oligonucleotides, exon skipping, and proteins was observed, except in heart, wherein high oligonucleotide levels but low exon skipping and dystrophin expression were seen. Overall, these results enhance our understanding of the pharmacokinetics and pharmacodynamics of 2'-O-methyl phosphorothioate oligos used for the treatment of DMD.Molecular Therapy-Nucleic Acids (2014) 3, e148; doi:10.1038/mtna.2014.1; published online 18 February 2014.
    Molecular Therapy - Nucleic Acids 02/2014; 3(2):e148. DOI:10.1038/mtna.2014.1 · 4.51 Impact Factor
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    • "It is also unknown if there are major differences between the uptake of PMOs and PPMOs into undifferentiated and differentiated skeletal muscle cells, i.e. myoblasts and myotubes, that would be responsible for the often uneven restoration of dystrophin in different muscle fibres. Furthermore, heart muscle always seems to represent a more challenging barrier to achieve efficient dystrophin correction, as naked PMOs are only weakly active at high repeated doses in this tissue (29), whereas PPMO conjugates are more active but give less activity in heart than in skeletal muscle (27,28). "
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    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.
    Nucleic Acids Research 12/2013; 42(5). DOI:10.1093/nar/gkt1220 · 9.11 Impact Factor
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    • "Phosphorodiamidate morpholino oligomers (PMOs) are currently one of the effective types of AOs for inducing exon-skipping in DMD patients.7 Systemic intravenous delivery of PMOs has been shown to restore dystrophin expression in multiple peripheral muscles in the dystrophin-deficient mdx mouse, which contains a premature stop codon in exon 23.3 However, much like other chemistries utilized,2,8 systemic correction was of relatively low efficiency and in the case of PMOs required a multiple-dosing regimen comprising seven weekly doses of 100 mg/kg3 or a single large dose of 3 g/kg9 with no or limited effect in heart. "
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    ABSTRACT: We have recently reported that cell-penetrating peptides (CPPs) and novel chimeric peptides containing CPP (referred as B peptide) and muscle-targeting peptide (referred as MSP) motifs significantly improve the systemic exon-skipping activity of morpholino phosphorodiamidate oligomers (PMOs) in dystrophin-deficient mdx mice. In the present study, the general mechanistic significance of the chimeric peptide configuration on the activity and tissue uptake of peptide conjugated PMOs in vivo was investigated. Four additional chimeric peptide-PMO conjugates including newly identified peptide 9 (B-9-PMO and 9-B-PMO) and control peptide 3 (B-3-PMO and 3-B-PMO) were tested in mdx mice. Immunohistochemical staining, RT-PCR and western blot results indicated that B-9-PMO induced significantly higher level of exon skipping and dystrophin restoration than its counterpart (9-B-PMO), further corroborating the notion that the activity of chimeric peptide-PMO conjugates is dependent on relative position of the tissue-targeting peptide motif within the chimeric peptide with respect to PMOs. Subsequent mechanistic studies showed that enhanced cellular uptake of B-MSP-PMO into muscle cells leads to increased exon-skipping activity in comparison with MSP-B-PMO. Surprisingly, further evidence showed that the uptake of chimeric peptide-PMO conjugates of both orientations (B-MSP-PMO and MSP-B-PMO) was ATP- and temperature-dependent and also partially mediated by heparan sulfate proteoglycans (HSPG), indicating that endocytosis is likely the main uptake pathway for both chimeric peptide-PMO conjugates. Collectively, our data demonstrate that peptide orientation in chimeric peptides is an important parameter that determines cellular uptake and activity when conjugated directly to oligonucleotides. These observations provide insight into the design of improved cell targeting compounds for future therapeutics studies.
    Molecular Therapy - Nucleic Acids 09/2013; 2(9):e124. DOI:10.1038/mtna.2013.51 · 4.51 Impact Factor
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