K. Honeyman

Dystrophin expression in the mdx mouse after localised and systemic administration of a morpholino antisense oligonucleotide.

Duchenne and Becker muscular dystrophies are allelic disorders arising from mutations in the dystrophin gene. Duchenne muscular dystrophy is characterised by an absence of functional protein, while Becker muscular dystrophy is usually caused by in‐frame deletions allowing synthesis of some functional protein. Treatment options are limited, and we are investigating the potential of transcript manipulation to overcome disease‐causing mutations. Antisense oligonucleotides have been used to induce specific exon removal during processing of the dystrophin primary transcript and thereby by‐pass protein‐truncating mutations. The antisense oligonucleotide chemistry most widely used to alter pre‐mRNA processing is 2′‐O‐methyl‐modified bases on a phosphorothioate backbone.

Improved antisense oligonucleotide induced exon skipping in the mdx mouse model of muscular dystrophy

Duchenne muscular dystrophy (DMD) is a fatal genetic disorder caused by dystrophin gene mutations that preclude synthesis of a functional protein. One potential treatment of the disorder has utilised antisense oligoribonucleotides (AOs) to induce removal of disease‐associated exons during pre‐mRNA processing. Induced in‐frame mRNA transcripts encode a shorter but functional dystrophin. We have investigated and improved the design of AOs capable of removing exon 23, and thus the disease‐causing nonsense mutation, from mRNA in the mdx mouse model of DMD.

Snapback SSCP analysis: Engineered conformation changes for the rapid typing of known mutations

Several approaches may be applied to detect known mutations, including restriction enzyme cleavage, allele‐specific oligonucleotide (ASO) hybridization or amplification, dideoxy fingerprinting, and direct DNA sequencing. All these approaches require several extra steps after PCR and may involve radioactive isotopes, time‐consuming hybridization, template purification, or digestion steps. The ease and simplicity of the SSCP test make it a popular choice for mutation detection, but a significant limitation is that some DNA changes will not alter the overall conformation of either single strand and are thus not amenable to SSCP typing. We describe Snapback SSCP to genotype normal and mdx mice (an animal model of Duchenne muscular dystrophy) that previously could not be differentiated by conventional SSCP analysis. A snapback primer was designed with additional bases at the 5′ terminus, which were complementary to the normal sequence flanking the mdx mutation and used under the original amplification conditions. Each single strand of these snapback PCR products now had one terminus capable of re‐annealing or “snapping back” to the normal sequence but not the mdx mutation. In this manner, a conformation change was engineered into the normal strand that could be readily distinguished from the mdx allele on a SSCP gel. This approach could be applied to the routine screening of other known mutations that are not amenable to detection by simple SSCP analysis. Hum Mutat 11:252–258, 1998.