Objective Duchenne muscular dystrophy (DMD) is caused by the inability to produce dystrophin protein at the myofiber membrane. A method to rescue dystrophin production by antisense oligonucleotides, termed `exon-skipping', has been reported for the mdx mouse and in four DMD patients by local intramuscular injection. We sought to test efficacy and toxicity of intravenous oligonucleotide (morpholino) induced exon skipping in the DMD dog model. Methods We tested a series of antisense drugs singly and as cocktails, both in primary cell culture, and two in vivo delivery methods (intramuscular injection, and systemic intravenous injection). The efficiency and efficacy of multi-exon skipping (exons 6-9) was tested at the mRNA, protein, histological, and clinical levels. Results Weekly or biweekly systemic intravenous injections with a three morpholino cocktail over the course of 5–22 weeks induced therapeutic levels of dystrophin expression throughout the body, with an average of about 26% normal levels. This was accompanied by reduced inflammatory signals examined by MRI and histology, improved or stabilized timed running tests and clinical symptoms. Blood tests indicated no evidence of toxicity. Interpretation This is the first report of widespread rescue of dystrophin expression to therapeutic levels in the dog model of DMD. This study also provides a proof of concept for systemic multi-exon skipping therapy. Use of cocktails of morpholinos, as shown here, allows broader application of this approach to a greater proportion of DMD patients (90%), and also offers the prospect of selecting deletions that optimize the functionality of the dystrophin protein.

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The B-cell leukaemia/lymphoma-2 (bcl-2) proto-oncogene is unusual as its product appears to provide survival advantage to B cells by blocking apoptosis. In this study, the expression of bcl-2 has been examined in normal non-haematopoietic tissues, embryos, and psoriatic skin by immunohistochemical staining. Bcl-2 protein expression is mainly observed in cell populations with a long life and/or proliferating ability such as duct cells in exocrine glands, basal keratinocytes, cells at the bottom of colon crypts, and neurons. In the skin of both adult and embryo and also embryonic kidney and cartilage, bcl-2 expression was observed in cells which were undergoing morphological transition from undifferentiated stem cells to committed precursor cells. The finding of bcl-2 expression in the terminal differentiated syncytial trophoblast, but not cytotrophoblast, and in some cells responsive to hormone stimulation such as in the endometrium and myometrium suggests that the gene expression may be related to hormone responsiveness. As no bcl-2 localization was seen in the benign hyperproliferative skin condition psoriasis, this does not suggest a straight-forward link to proliferation. These observations support the view that the bcl-2 gene may have an important role in cell development, maturation, and the path to terminal differentiation.

Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy, arising from mutations in the dystrophin gene that preclude the synthesis of functional protein. Antisense oligonucleotides (AOs) have been shown to induce specific exon skipping and thereby restore the reading frame and expression of functional dystrophin. In this report, we examine the effects of peptide nucleic acid (PNA) oligonucleotides and PNAs conjugated with peptides including TAT, muscle-specific peptide (MSP), adeno-associated virus 6 (AAV6) functional domain (AAV6), and AAV8 functional domain (AAV8), on exon skipping in vitro and in vivo. Efficient skipping of targeted exon 23 was achieved in cultured mdx myoblasts with PNA and PNA-peptide conjugates. Furthermore, single intramuscular injections of PNA and all PNA-peptide conjugates resulted in significant numbers of dystrophin-positive fibers in the injected tibialis anterior (TA) muscles of mdx mice, with no apparent local toxicity. Similar effects of exon skipping and dystrophin expression were obtained in mice of all ages. PNA and PNA-AAV6, PNA-AAV8 conjugates induced dystrophin expression in a dose-dependent manner. Our results demonstrate that PNAs have a higher efficiency of exon skipping than 2'O methyl phosphorothioate AOs do, and have a potential use in AO chemistry for antisense therapy of DMD.