Cryptic splicing involving the splice site mutation in the canine model of Duchenne muscular dystrophy

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Abstract

Golden retriever muscular dystrophy arises from a mutation in the acceptor splice site of intron 6 of the dystrophin gene. Skipping of exon 7 disrupts the mRNA reading frame and results in premature termination of translation. We are using this animal model to evaluate treatments for Duchenne muscular dystrophy, including gene repair induced by chimeric oligonucleotides. After injection of golden retriever muscular dystrophy (GRMD) muscle with a chimeric oligonucleotide to repair the lesion, immunostaining revealed a modest increase in the number of dystrophin-positive fibres at the injection sites. Dystrophin gene transcripts containing exon 7 were detected by reverse transcription-polymerase chain reaction, suggesting that low levels of splice site correction may have occurred. However, DNA sequencing of these apparently normal dystrophin gene transcripts revealed that the first five bases of exon 7 were missing. It will be important to be aware of this phenomenon with respect to further gene correction studies in the canine model.

Introduction

Golden retriever muscular dystrophy (GRMD) [1] has been used as a model to study therapeutic intervention for Duchenne muscular dystrophy (DMD). We have attempted gene replacement using a first generation recombinant adenovirus with the dystrophin minigene [2]. Although initial experiments were promising, the limitations of inefficient delivery and lack of sustained transgene expression were encountered. The immune response to both the dystrophin gene product and viral antigens was found to be an additional obstacle.

The dystrophin gene could be regarded as one of the greatest challenges to gene therapy. Not only is it the largest gene known, but it is under tissue specific control with numerous promoters both 5′ to the gene and internally [3], [4], [5]. Efficacy of the conventional gene replacement approach could be further compromised as only the skeletal muscle isoform can be delivered at any one time with the current viral vectors. The role of the non-muscle isoforms is not known [6].

Approximately two thirds of DMD mutations arise from the deletion of one or more exons [7]. The remaining cases arise from more subtle DNA rearrangements, typically non-sense mutations or splice site mutations that disrupt the translational reading frame of the dystrophin mRNA [8], [9].

The possibility of DNA repair of point mutations provides a tantalizing prospect of therapy for a significant proportion of DMD cases. Successful correction or repair of point mutations through the use of chimeric oligonucleotides [10], [11], [12], [13] or short fragment homologous recombination [14], [15] could circumvent many of the problems besieging gene replacement.

Many groups have reported significant variation in efficacy or levels of gene correction and raised some serious concerns about chimeric oligonucleotides as a viable therapy. Individual laboratories (and even the same researcher) have reported gene correction efficiency may vary from 0 to 30% (for comment see Ref. [16]). However, continuing reports from different laboratories on gene correction of a variety of targets suggest that this approach has great potential [10], [11], [12], [13]. Clearly there are many variables and parameters that have yet to be fully understood and appreciated.

We approached gene correction in the GRMD model with caution and sought to include as many controls as possible to ensure the validity of the results. Consequently we identified a relatively rare dystrophin mRNA that contained all but the first five bases of exon 7. This transcript had arisen from the selection of the first AG in exon 7 as an alternate splice acceptor site. This phenomenon of exon 7 inclusion in GRMD dystrophin transcripts has not been previously reported in dystrophic canine muscle and has the potential to compromise gene correction studies. This transcript is out of frame and cannot be responsible for the origin of revertant fibres.

Section snippets

Animals and chimeric oligonucleotide delivery

A litter of ten pups, born to a GRMD carrier bitch inseminated with semen from an affected male, was genotyped [17] to identify three affected pups. Animals were housed and cared for at the Murdoch University Veterinary School Animal facility.

The chimeric oligonucleotide CO-GRMD7G>A, supplied by Integrated DNA Technologies (Coralville, IA) was high-performance liquid chromatography (HPLC) and gel purified by the supplier. The sequence is shown with the 2′O-methyl bases indicated in lower case,

Immunostaining of CO-GRMD7G>A treated dystrophic muscle

Only three affected pups were available for this experiment, thus numbers of injected sites and parameters were limited. Sections from uninjected sites typically contained 0–1 dystrophin positive fibres while sections from all the injected sites had 1–5 dystrophin positive fibres. Our currently available antibodies could not distinguish normal canine dystrophin from revertant canine dystrophin. As we were aware that dystrophin observed at the injection site could have arisen from revertant

Discussion

We used chimeric oligonucleotides in an attempt to correct the point mutation responsible for GRMD. Conscious of some concerns regarding this approach, we were aware of the need for stringent controls. Initial experiments were performed in the absence of normal canine tissue (or nucleic acid) that could have been a potential source of contamination.

Initial immunohistochemical studies suggested low levels of correction may have taken place as there was a very modest increase in the number of

Acknowledgements

This work was supported by the Muscular Dystrophy Association of the United States of America, the Medical Research Foundation of Western Australia and the Neuromuscular Foundation of Western Australia. The authors wish to thank Linda Davies, Andy Cheng and Frances Lloyd for expert technical assistance.

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