The following three abstracts describe Morpholino experiments toward development of a Duchenne muscular dystrophy treatment and will be presented at the 13th International Congress of the World Muscle Society, Newcastle, September 2008.

Original web page (with all abstracts):
http://www.distrofiamuscular.net/abstracts19.htm

3) Dystrophin expression after systemic delivery of morpholino antisense oligonucleotide in mdx mouse: A dose–response analysis

A. Malerba(a), G. Dickson(a) and I. Graham(a)

(a)Centre for Biomedical Sciences, University of London, Royal Holloway, Egham, Surrey, United Kingdom

Duchenne muscular dystrophy (DMD) is the most common congenital muscular disorder. It is caused by mutations that create premature termination of dystrophin translation. One of the most promising approaches for the treatment of DMD is the administration of antisense oligonucleotide (AO). This approach applied to human muscle cells and in animal models of DMD restored the correct reading frame in the dystrophin transcript, thereby producing a shorter but partially functional protein. One of the most widely used AO chemistries is the phosphorodiamidate morpholino oligomer (morpholino). While it has been clearly demonstrated that administration of morpholino to mdx mice allows the skipping of the mutated exon 23, little is known about the pharmacokinetics and the best doses for the administration of this AO. It has been demonstrated that several injections of morpholino induce the expression of dystrophin in more fibres than after a single injection, but no data have been published that compare the administration of the same total dose over a set period of time using different numbers of administrations or different routes. Here we report that multiple injections of a low amount of morpholino show significantly more dystrophin positive fibres in a wide variety of muscle groups 8 weeks after administration than a single high dose with the total amount. Other histological features, such as the cross sectional area, the centronucleation index and the expression of the dystrophin-associated protein complex, showed a significant improvement in mice treated by repeated injection. These results show that it is feasible to restore dystrophin expression with high efficiency by repeated injections of low doses of morpholino.

4) Induced exon skipping in normal and mdx muscle

S. Fletcher(a), J.P. Steinhaus(a), C. Mitrpant(a), P.L. Meloni(a) and S.D. Wilton(a)

(a)Centre for Neuromuscular & Neurological Disorders, University of Western Australia, Perth, Australia

Induced exon skipping to remove or by-pass protein truncating mutations in the dystrophin gene is emerging as a potential therapy for many cases of Duchenne muscular dystrophy. It has been proposed that the compromised sarcolemma of the dystrophic muscle fibres may facilitate uptake of compounds that induce exon skipping. If this were the case, then restoration of some functional dystrophin expression may restrict further oligomer uptake, thereby creating a therapeutic ceiling. We addressed this question by systemically treating normal C57BL/10ScSn mice with the same compound that induced substantial dystrophin exon 23 skipping and restored dystrophin expression in the mdx mouse model of muscular dystrophy (C57BL/10ScSnmdx). Repeated intraperitoneal injections of a phosphorodiamidate morpholino oligomer coupled to a cell penetrating peptide (PMO-P007), were sufficient to induce readily detectable levels of dystrophin gene transcripts missing exon 23 in normal skeletal muscle, as detected by RT-PCR. However, exon 23 skipping could not be detected in the heart until assay conditions were biased towards generation of shorter PCR products, after which 22% exon skipping was apparent in cardiac muscle from treated animals. Detailed protein studies were not possible on the normal dystrophin-positive background, but clearly, the uptake and efficacy of PMO-P007 was not compromised by the normal skeletal muscle sarcolemma. Furthermore, the selective bias that can be achieved to enhance apparent exon skipping during RT-PCR assays was such that we recommend molecular testing should be standardised to facilitate valid comparisons between different laboratories and studies.

5) Restoration of dystrophin expression in mdx mouse by peptide-conjugated antisense oligonucleotide

H.F. Yin(1), Y.Q. Seow(2), H.M. Moulton(3), P.L. Iversen(3), J.K. Boutilier(3) and M.J.A. Wood(2)

(1)Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom (2)University of Oxford, Oxford, United Kingdom (3)AVI BioPharma Inc., Corvalis, United States

Duchene muscular dystrophy (DMD) is a severe muscle wasting disease caused by mutations in the dystrophin gene. The efficacy of antisense oligonucleotide (AO)-mediated exon skipping for the restoration of dystrophin has been established in animal models and in DMD patients. However there remain significant limitations to this therapeutic approach due to the lack of effective systemic AO delivery to muscle. Here we investigate systemic muscle-specific AO delivery by testing AOs directly conjugated to cell penetrating peptides (CPPs) or to tissue-specific homing peptides (e.g. muscle-specific peptide, MSP). Morpholino chemistry AOs were directly conjugated to CPPs or to homing peptides and evaluated in mdx mice following systemic delivery. Effective exon skipping and dystrophin expression were induced in body-wide skeletal muscles and in cardiac muscles. This is the first time that the successful body-wide restorations of dystrophin expression even in heart have been achieved at low AO doses. In parallel we also report the discovery and characterization of a novel delivery formulation which facilitates AO uptake in muscle. A series of studies have shown that this delivery formulation enhances the delivery of AOs of different chemistry (e.g. 2-OMeRNA, PNA and morphlino), depends on the activity of specific muscle membrane transporters, and that it induces significant restoration of dystrophin expression in muscle compared with commonly used delivery formulations. In summary, we report data demonstrating the potential of muscle-specific homing peptides, CPPs and novel delivery formulations for the targeted restoration of dystrophin in DMD.