Pre-mRNA splicing manipulation via Antisense Oligomers

Duchenne muscular dystrophy (DMD), the most common lethal neuromuscular disease in childhood, arises from protein-truncating mutations in the dystrophin gene. A deficiency in dystrophin leads to loss of the dystrophin associated protein complex (DAPC), which in turn, renders muscle fibres vulnerable to injury, and eventually leads to muscle loss, necrosis and fibrosis. Although, the dystrophin gene was identified nearly two decades ago, and extensive research has been directed at finding a therapy for DMD, to date, there is still no effective treatment available. One promising molecular approach to treat DNID is antisense oligomer (AO) induced splice intervention. AOs were most widely used to induce RNaseH-mediated gene transcript degradation, however, the development of different backbone chemistries heralds a new generation of AOs that can modify gene transcript splicing patterns. Application of AOs to the dystrophin pre-mRNA to influence exon selection and induce shortened, in-frame dystrophin isoforms is being vigorously pursued. The majority of the work presented here explores the concept of personalised therapies for DMD, whereby oligomers are designed to specifically target individual mutations. The importance of AO-optimisation to obtain AOs capable of inducing efficient dual exon skipping in an established animal model of muscular dystrophy (4CV mouse), which carries a DMD-causing mutation in exon 53, is demonstrated. Removal of both exons 52 and 53 was required to by-pass the mutation, maintain the reading frame and restore dystrophin expression. One of the major challenges of AO-induced splice intervention for therapeutic purposes will be the design and development of clinically relevant oligomers for many different mutations. Various models, including cells transfected with artificial constructs and mice carrying a human dystrophin transgene, have been proposed as tools to facilitate oligomer design for splice manipulation. This thesis investigates the relevance of using mouse models to design AOs for human application, and also explores the use of cultured human myoblasts, from both unaffected humans and a DMD patient, as a means of establishing the most effective therapeutic compound. In addition to induction of exon skipping, the applicability of AOs to promote exon inclusion, by masking possible intronic silencing motifs of survival motor neuron (SMN) pre-mRNA in cultured fibroblasts from a spinal muscular atrophy (SMA) patient, is investigated. This study provides additional information about a novel oligomer target site that could be used in combination with previously identified splice silencing motifs for a molecular therapeutic approach to SMA, and may perhaps open up new avenues of treatment for other genetic disorders, where oIigomers could be used to induce exon inclusion