Quantification of a serum titin fragment reflects dystrophin restoration in mdx mice and disease severity in patients with dystrophinopathies

Increased dystrophin expression in muscle has been accepted as a surrogate endpoint for accelerated approval of gene therapies within Duchenne Muscular Dystrophy (DMD). Previous studies identified an N-terminal titin fragment in urine as a biomarker suitable for monitoring disease progression and therapeutic interventions. Plasma levels of a titin fragment comprising aa 2,229–2,352 were found to negatively correlate with dystrophin expression in mdx mice treated with AAV9-CK8-μDys5 micro-dystrophin gene therapy. In this study, we sought to investigate which titin fragments can be detected in serum from patients with DMD and Becker muscular dystrophy (BMD). Using bottom-up selected reaction monitoring tandem mass spectrometry, we identified several serum peptides originating from the central region of titin. Six monospecific antibodies targeting this fragment could recognise a proteolytic titin product at 100 kDa, present in serum from DMD and BMD patients, but not healthy controls. This fragment showed a steeper age-related decline in patients with DMD compared to those with BMD. We further developed a quantitative sandwich immunoassay and showed that the concentrations of this fragment were between 130 and 2,390 pM in DMD patients and 110 and 3,970 pM in BMD patients. The same assay showed titin concentration, between 170 and 990 pM in plasma from treated mdx mice, that had a significant Pearson correlation of -0.76 ( P =1.53e−08) with (micro)dystrophin expression in quadriceps. If the negative correlation with dystrophin expression in muscle is also confirmed in DMD patients treated with micro-dystrophin therapies, this serum titin fragment may serve as a potential pharmacodynamic biomarker.