169PComparison of western-blot, mass spectrometry and simple-western methods shows that simple-western is the most sensitive method to detect µ-dystrophin

Duchenne muscular dystrophy (DMD), a rare X-linked genetic disorder affecting male children, leads to progressively severe muscle dysfunction often associated with respiratory and/or cardiac insufficiencies and premature death. The pathology is caused by out-of-frame mutations in the DMD gene, leading to absence of dystrophin, a large muscle protein. As the large size of dystrophin cDNA impedes its packaging in AAV vectors, various shorter micro-dystrophin (µDys) transgenes are used for clinical development. Different analytical methods may be used to measure the µDys levels from muscle biopsies. GNT-016, a clinical trial investigating an AAV-based µDYS, uses the simple-western (SW) method for quantifying the therapeutic product. In an effort to document the differences in methodologies, we performed a side-by-side comparison of 3 methods used in DMD clinical trials. Total proteins extracted from DMDmdx rats’ muscles sampled after intravenous treatment with an AAV8-spC5-12-µDYS were used for the quantification of µDYS in dose-response experiments performed by nano LC/MS mass spectrometry, western-blot and SW methodologies. SW is a capillary-based electrophoretic method allowing separation and detection of proteins in a single device. Both mass spectrometry and SW prove advantageous compared to western-blot in terms of reproducibility and repeatability (CV<25% for SW and CV<8% for mass spectrometry). However, SW is over 4,000 times more sensitive than the 2 other methods. Considering the limited amounts of patient’s tissue available to measure dystrophin quantities, sensitivity appears to be a key factor. SW variability is lower than the recommended guidelines and easier to set-up than mass spectrometry, which requires very specific equipment and technical expertise. This method, used for the quantification of µDYS expression in our clinical trial, was globally consistent with the immunohistological results performed on the same samples.