source: American Academy of Neurology
FJ Schnell, C Donoghue, J Dworzak, JS Charleston, DE Frank, S Wilton, S Lewis, JR Mendell, LR Rodino-Klapac, Z Sahenk
Phosphorodiamidate morpholino oligomer (PMO) eteplirsen, which received accelerated approval in the US for patients with DMD gene mutations amenable to exon 51 skipping, along with other oligonucleotides under investigation in late-stage clinical trials, aim to enable production of internally-deleted dystrophin protein.
DMD is primarily caused by whole-exon deletions resulting in a shift of the mRNA reading frame that prevents production of functional dystrophin protein. Western blotting has been traditionally used as the confirmatory assay for clinical diagnosis of DMD or Becker Muscular Dystrophy (BMD), but the methods are not standardized and lack true quantitative capacity.
A sensitive and semi-quantitative Western blot method was developed to detect dystrophin in human muscle biopsies utilizing a standard curve composed of non-DMD/BMD (normal control) muscle lysate spiked into DMD muscle lysate to maintain equivalent protein load. Running a 5-point standard curve ranging from 0.25% – 4% of a single normal control on every gel allows for calculation of dystrophin levels for unknown samples run on the same gel and enables normalization of slight fluctuations in dystrophin signal intensity between gels. Fixed film exposure times ensure dystrophin signal is not oversaturated and remains within the dynamic range of the assay.
In an interim analysis following 48 weeks of treatment in a Phase III clinical study of eteplirsen, 12 biopsied patients showed a significant mean dystrophin increase by Western blot following treatment compared to their pre-treatment biopsies (P<0.05). This method allowed for confirmation of mean increases in dystrophin production following eteplirsen-treatment which were the basis of eteplirsen accelerated approval.
The Western blot method was validated according to FDA Draft Guidance for Industry, Bioanalytical Method Validation, and can provide confirmation of mechanism of action for dystrophin restoring therapies. The developed method enables sensitive and robust quantification of dystrophin down to 0.25% of “normal” levels.
Sarepta Therapeutics Inc., USA; Murdoch University, Australia; Nationwide Children’s Hospital, USA