Steve Wilton AO FAHMS BSc PhD

Duplications of one or more dystrophin exons that disrupt the reading frame account for about 15% of all Duchenne cases, and like the more common genomic deletions, most pathogenic duplications of single or multiple dystrophin exons are also amenable to targeted exon skipping. However, additional considerations must be taken into account: (i) Skipping of all duplicated exons, and flanking exons as necessary, will frequently be required to restore the reading frame and generate an in-frame Becker muscular dystrophy-like mRNA, (ii) the phosphorodiamidate morpholino oligomer chemistry is more effective than the 2′-O-methyl modified oligonucleotides at inducing multiple exon skipping, and (iii) the apparent efficiency of exon skipping can be confounded by the choice of RT-PCR system. Standard RT-PCR systems can preferentially amplify the shorter amplicons, implying more efficient exon skipping than may actually be induced. Unless high fidelity RT-PCR systems are used, strand slippage during annealing/elongation steps will generate normal length transcripts that are artifacts of the amplification.

The pre-mRNA splicing process is an essential aspect of gene expression and function and plays a substantial role in the complexity of higher eukaryotes. The development of antisense oligonucleotides (AOs) to harness the splicing process and manipulate it to treat various inherited and acquired diseases has been boosted by its flexibility and customisation capability. As the amount of research in this space increases, certain aspects need to be considered, in particular, how nonsequential splicing of pre-mRNA can impact AO-mediated splicing manipulation. In this chapter, we reviewed literature discussing intron removal order and several examples of disease-causing mutations impacted by this phenomenon. We also compared two strategies used to study intron removal order and the occasions that they are best suited. Finally, we discuss how nonsequential splicing could facilitate or impede the development of splice-manipulating AOs and aspects to consider when analysing AO effectiveness.

Duplications of one or more dystrophin exons that disrupt the reading frame account for about 15% of all Duchenne cases, and like the more common genomic deletions, most pathogenic duplications of single or multiple dystrophin exons are also amenable to targeted exon skipping. However, additional considerations must be taken into account: (1) skipping of all duplicated exons, and, flanking exons as necessary, will frequently be required to restore the reading frame and generate an in-frame Becker muscular dystrophy-like mRNA, (2) the phosphorodiamidate morpholino oligomer chemistry is more effective than the 2′-O-methyl modified oligonucleotides at inducing multiple exon skipping, and (2) the apparent efficiency of exon skipping can be confounded by the choice of RT-PCR system. Standard RT-PCR systems can preferentially amplify the shorter amplicons, implying more efficient exon skipping than may actually be induced. Unless high fidelity RT-PCR systems are used, strand slippage during annealing/elongation steps will generate normal length transcripts that are artifacts of the amplification.

Nucleic acid aptamers have attracted considerable attention as tools for targeted molecular recognition. This chapter describes advances in aptamer technology in the field of neurological diseases. Aptamers possess certain advantages over antibodies, such as stability, production scale, method and time, chemical fabrication, and costs. Multiple sclerosis is a chronic inflammatory and demyelinating disorder of the nervous system. A combination of these aptamers with other therapeutics payload might improve the overall treatment efficacy of glioblastoma cells significantly. Thrombolysis-related hemorrhage is one of the major obstacles in the development of stroke treatments, because new therapeutics needs to be immediately reversible in case of hemorrhage occurrence. Parkinson disease is a neurodegenerative disorder of the central nervous system, mainly affecting the motor neuron system. Alzheimer disease is the most common neurodegenerative disease and is accountable for more than 80% of all cases of dementia in the elderly. Myasthenia gravis is a disease that interferes with the signal transduction between neurons and muscle cells.

We have taken an empirical approach in designing splice-switching oligomers to induce targeted dystrophin exon skipping. The nucleotide sequence of the exon under examination is first analyzed for potential exon recognition motifs and then a set of oligomers complementary to the acceptor and donor splice sites, as well as intra-exonic regions predicted to contain exon splice enhancers, are designed and synthesized as 2′-O-methyl-modified bases on a phosphorothioate backbone (2OMeAOs). The 2OMeAOs can be readily transfected into cultured normal myogenic cells as cationic lipoplexes, and are incubated for 24 h before total RNA extraction and subsequent analysis by semi-quantitative RT-PCR. The amplification conditions used for each dystrophin transcript region under investigation minimize preferential production of shorter amplicons and do not exaggerate the level of induced RT-PCR products, compared to the endogenous dystrophin transcript product. It is imperative that the test oligomers are transfected over a range of concentrations and that the target exon is excised in a reproducible and dose-dependent manner. Once it has been demonstrated that an oligomer can induce some degree of exon skipping, that target region of the pre-mRNA is assumed to be involved in splicing of the exon. A series of overlapping oligomers are prepared and evaluated by transfection into normal myogenic cells at lower concentrations to identify the more effective compounds. Clinical application requires antisense compounds that efficiently modulate splicing at low dosages, delivering the greatest benefits in terms of efficacy, safety, and cost.