Chalermchai Mitrpant

Duchenne muscular dystrophy (DMD) is a progressive, fatal muscle wasting disorder with a predictable course and limited treatment options. Advances in clinical care and management have almost doubled the life expectancy of affected boys over the last 2–3 decades, but do not address the primary etiology of DMD, the loss of dystrophin. Corticosteroids are effective in stabilizing muscle strength and prolonging ambulation, although the exact mechanism by which steroids slow the dystrophic process is unknown. Despite the limited therapeutic value of corticosteroids these drugs represent the best treatment option currently available, and a large proportion of DMD patients are treated with prednisone/prednisolone or deflazacort. Biological therapeutics are becoming available, and it will be important to establish whether these compounds can be safely administered to patients being treated with corticosteroids. Antisense oligomer-mediated splicing manipulation can bypass dystrophin gene lesions and is showing promise as a therapy for DMD. Intramuscular injection of RNA analogues, targeting exon 51, in DMD patients has resulted in specific exon exclusion and dystrophin expression in the treated muscle. We report that concurrent peptide-conjugated phosphorodiamidate morpholino oligomer and prednisolone administration is not contraindicated in mdx mice and that muscle physiology is improved by the combined treatment.

Spinal muscular atrophy (SMA) is the most common autosomal recessive neurodegenerative disorder of children with an incidence of 1 in 10,000 live births and a carrier frequency of 1 in 40-50 adults. SMA is attributable to a deficiency in the survival of motor neuron protein (SMN) caused in most patients by mutation of the SMN1 gene. Deficiency of SMN protein results in degeneration of anterior horn cells leading to hypotonia, symmetrical muscle weakness, fasciculation of the tongue muscles and tremors of the fingers and hands. There are two genomic copies of the SMN gene (SMN1 and SMN2) and expression of the full length SMN2 gene product has been shown to partly compensate for the lack of SMN1 product. However, a single base difference (C/T) at the sixth nucleotide in exon 7 of the SMN2 gene promotes excision of that exon from the mature transcript leading to production of only a minimal amount of full-length protein. The promotion of exon 7 inclusion in the SMN2 transcript by masking splice silencing motifs with antisense oligonucleotides (AO) is a potential intervention to increase the level of full-length SMN protein. Using a panel of modified 2'-O-methyl AO’s (phosphorothioate backbone) targeted across exon 7 of the SMN2 pre-mRNA we aimed to identify possible splice silencing motifs that would lead to exon 7 inclusion during SMN2 expression in SMA fibroblasts. While our results failed to demonstrate a strong exonic silencing motif which could be masked to promote exon 7 inclusion parallel experiments in normal control cells showed robust exon 7 skipping could be induced by AO’s targeted near the exon 7 donor splice site. Therefore, this study has provided additional insight into exon splicing as it relates to SMN exon 7 providing a potential model with which to study the functionality of alternatively spliced SMN proteins.

Clinical trials are underway to demonstrate that antisense oligomers (AOs) can redirect dystrophin gene transcript splicing to excise selected exons, and thereby remove protein truncating mutations that would otherwise lead to Duchenne muscular dystrophy (DMD). Due to the widespread and complex nature of dystrophin gene expression, DMD is a great challenge to any therapy. However, characterization of the gene structure in Becker MD patients presenting with mild phenotypes, indicate that substantial portions of the dystrophin gene can be lost with relatively minor consequences, and some in-frame deletions may only be identified late in life. It as been confirmed that over half of the 79 exons are redundant, when lost in particular combinations. While 10-12 AOs should restore the reading- frame in the more common genomic deletion hotspots, scores of AOs will be needed to by-pass the many different protein-truncating mutations spread across the gene. The immediate challenges are (i) to establish effective dosage regimens, (ii) gain acceptance for these personalized genetic medicines as class-specific compounds, (iii) extend the treatment to all potentially amenable mutations in the dystrophin gene and (iv) apply this platform to other acquired and genetic conditions.

Duchenne Muscular dystrophy (DMD), a severe neuromuscular disorder, is caused by nonsense or frameshift mutations in dystrophin gene that results in an absence of functional dystrophin. Loss of functional dystrophin renders muscle fibres vulnerable to membrane damage during contraction. Antisense oligonucleotide (AO) induced exon skipping has been used to induce specific exon removal and by-pass the disease-causing mutation. Some dystrophin mutations will require removal of more than one exon. We are investigating multi-exon skipping in the B6Ros.Cg-Dmdmdx−4Cv/J (4cv) muscular dystrophy mouse model, which has a nonsense mutation in exon 53 of the dystrophin gene. This area is of relevance to the human dystrophin gene since this area corresponds to the major deletion hot spot of the human dystrophin gene. To restore the reading frame of the 4cv dystrophin mRNA, both exons 52 and 53 must be excised from the mature dystrophin gene transcript. 2_-O-Methyl AOs on a phosphorothioate backbone (2OMe) have been designed to mask motifs involved in splicing to remove these exons during pre-mRNA processing. We describe the removal of single exon and multi-exons in cell culture and in vivo. The exploration of exon skipping events in the 4CV mouse model will provide additional information in AO design, and will be a molecular model relevant to many DMD cases.