Biochemical outcome measures

Comparison of exon skipping

Duchenne muscular dystrophy (DMD) patients have different genetic mutations in the DMD gene that encodes the dystrophin protein. Due to out-of-frame mutations of the DMD gene, the reading frame is shifted, resulting in a truncated, non-functional dystrophin protein in DMD patients’ muscles. Clinical trials for DMD are currently in progress using antisense oligonucleotide (AON)-mediated exon skipping. AONs target exons during dystrophin pre-mRNA splicing to hide it from the splicing machinery, and cause exon skipping to restore the reading frame, allowing the production of shorter but functional dystrophins.

Protocols to quantify exon skipping levels for e.g. clinical trials are not standardized yet, making it difficult to compare results from different research groups. The BOM working group is currently working to find a reliable and standardized method to assess exon skipping levels. The development of one common standardized operating procedure (SOP) will allow comparison of exon skipping results across different labs and clinical trials, and can be used as starting material for generating similar SOPs to study exon skipping efficiency for other genes.

Towards these aims, two DMD muscle cell lines (carrying deletions of exons 48-50 and of exon 52) will be transfected with an AON to skip exon 51. Skipping of exon 51 is in late stage development for DMD and is intended for approximately 13% of all DMD patients. Therefore, blinded RNA samples will be sent to the participating laboratories where exon skipping will be quantified. Cell culturing, AON transfections and RNA isolations will be done by one single lab to provide the same starting material to all participating laboratories. The inter-laboratory variability of the different protocols will be assessed and the results were presented and discussed at a BOM meeting in May 2015.

BOM working group meeting

The BOM working group meeting was held in Leiden, the Netherlands, on the 19^th of May 2015. Colleagues involved in exon skipping for Duchenne Muscular Dystrophy (DMD) from the University College London, the Royal Holloway University of London, the University of Ferrara, the Leiden University Medical Center, BioCruces and BioMarin participated in the meeting. One of the aims of the BOM working group was to find a reliable and standardized method to compare exon skipping levels among different research groups and samples of clinical trials. Methods to determine exon skipping levels and transcript instability have been compared.

Therefore, two DMD muscle cell lines (carrying deletions of exons 48-50 and of exon 52) were treated with an antisense oligonucleotide to skip exon 51 of the DMD gene. Transfection, RNA isolation and blinding of the samples were done by a single laboratory prior to share the samples with the other participating laboratories. Each laboratory distributed the protocol to determine exon skipping levels in house with the BOM working group partners. The protocols, which varied in technologies and reagents used, were replicated by the different laboratories. Results were presented at the meeting and the inter-laboratory reproducibility of the different methods was assessed. The strengths and weaknesses of the different protocols resulted in a fruitful discussion. The coding of the blinded samples was decrypted after all data were presented.

Work is in progress to finalize the analysis of the results and to provide a reliable and standardized method to the Duchenne community working on therapeutic exon skipping.

Working group leaders

Working group members

Future activities

Will be announced here in due course.

Comparing Dystrophin Quantification Methods

Exon skipping generally aims to restore the production of a missing protein. As such protein quantification is an important biochemical outcome measure to assess whether or not the therapy has worked. For Duchenne muscular dystrophy the exon skipping approach aims to restore dystrophin protein. However, protocols to quantify dystrophin are not standardized, making it difficult to compare results from different research groups.

The aim of the biochemical outcome measures (BOM) working group is to develop standardized operating procedures for dystrophin quantification, and also to train personnel in these procecures.

Notably, to quantify dystrophin invasive muscle biopsies are needed. Ideally, treatment response on a biochemical level would be assessed in tissues that are easier obtainable (e.g. serum or urine), through surrogate markers. A second aim of the BOM working group is to compare notes on promising candidate surrogate markers, to harmonize and optimize assays to measure them and to facilitate their validation for use in clinical trials.

Working group leaders

Working group members

Future activities

Will be announced here in due course.

Past activities

BOM working group meeting - London, UK - 26th March 2014

BOM working group meeting – Leiden, the Netherlands, 9th May 2015

One of the aims of the BOM working group is to develop standardized operating procedures for dystrophin quantification. If it can be shown that dystrophin can be reliably quantified by standardised methods, this would facilitate using dystrophin restoration as a pharmacodynamic outcome measure exon skipping trials in DMD. This is why several expert labs compared and standardised their protocols for dystrophin quantification prior to testing these protocols on muscle samples from Duchenne and Becker muscular dystrophy patients sent blindly to all of them. These samples expressed variable amounts of dystrophin. There was a good correlation between dystrophin levels assessed by different groups and there also was a good correlation between the two techniques used in the quantification (western blotting and immunohistochemistry). The results of this effort were recently published in Neurology - www.ncbi.nlm.nih.gov/pubmed/25355828