Proteomic strategies for blood biomarker development in rare dystrophinopathies

Sammanfattning: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are two rare genetic disorders of the family dystrophinopathy. They are both caused by the lack of, or reduced production of, the protein dystrophin. Due to abnormal dystrophin expression, patients experience progressive loss of muscle mass and cardio-, respiratory- and sometimes cognitive complications. DMD is the more severe form of dystrophinopathy, which manifests in young children and leads to wheelchair confinement in early teens followed by bed confinement and a shortened life expectancy. Dystrophin expression is absent or at less than 3% in DMD patients, often due to frame-shift mutations which cause protein expression to stop pre-maturely. BMD patients, on the other hand, display a higher but variable expression of dystrophin, often with large internal truncations. This partial expression results in a milder phenotype than DMD with sometimes unaffected life expectancies compared to healthy individuals. In the past decade, there has been substantial research into therapies aiming at increasing dystrophin expression in DMD patients and thereby prolonging ambulation, with the first gene-therapy gaining regulatory approval from the U.S. Food and Drug Administration (FDA) in June 2023. Current regulatory approvals for treatments of DMD patients have relied on dystrophin quantification in muscle biopsies as a biomarker and surrogate endpoint to predict a possible benefit from treatment, but these tests require repeated collection of muscle biopsies. Biomarkers are biochemical or physiological laboratory tests that measure a biological processes or condition. There is a need for monitoring biomarkers in dystrophinopathies, as well as biomarkers which can be used to predict outcome in clinical trials. As patients are often young children, it is important to develop biomarkers from less invasive and more readily available biological samples than muscle biopsies, such as blood or urine.In this thesis, we have used affinity proteomics and mass spectrometry to identify and validate biomarkers for monitoring disease progression in DMD and BMD patients from serum or plasma. The overall aim has been to identify biomarkers capable of distinguishing between patients with different levels of dystrophin expression and rates of disease progression in order to suggest gene-therapy pharmacodynamic biomarkers. In Paper I, we used suspension bead array (SBA) technology to identify biomarker candidates which reflect disease progression in DMD. Ten proteins were identified as related to disease progression. The ten biomarker candidates identified in Paper I were further analytically validated in Paper II using two orthogonal and absolute quantitative methods, parallel reaction monitoring mass spectrometry (PRM-MS) and sandwich immunoassays, which resulted in five analytically validated disease monitoring biomarkers for DMD (CA3, MYL3, LDHB, COL1A1 and FGG).Dystrophin-restoring therapies build on the hypothesis that increasing expression of internally deleted dystrophin reduces disease severity. In BMD patients, partial expression of short dystrophin molecules results in a milder phenotype than in DMD patients lacking expression of dystrophin. However, there are some differences in the nature of disease progression between DMD and BMD. In Paper III, we used Data Independent Acquisition Mass Spectrometry (DIA-MS) to study proteomic similarities and differences between DMD and BMD disease progression. This study revealed some discrepancies between disease progression biomarker candidates in the two related disorders.In Paper IV, we searched for blood biomarkers capable of reflecting changes in dystrophin expression during gene-therapy clinical trials. We identified ten proteins which correlated with dystrophin or microdystrophin expression in DMD mouse models. Out of these ten proteins, we identified that myosin light chain 3 (MYL3) declined steeper over time in dystrophinopathy patients with low or no dystrophin expression compared to patients with higher dystrophin expression. Two more biomarkers were identified in Paper IV as potentially related to dystrophin expression in muscle biopsies from both mouse models and patients. These were titin (TTN) and, interestingly, serum leakage of dystrophin. The possible presence of dystrophin in blood has not been well studied, and only one prior publication suggests that dystrophin may be a biomarker for DMD. Many DMD therapies currently in clinical trials aims at restoring dystrophin production and for those trials, the possibility of monitoring dystrophin leakage into blood could provide valuable information on therapeutic efficacy. In Paper V, we designed a proof-of-principle study to explore if dystrophin in blood can be a DMD biomarker. In conclusion, this thesis explores disease progression monitoring biomarkers and gene-therapy pharmacodynamic biomarkers for DMD and BMD. Three proteins, MYL3, TTN, and serum levels of dystrophin, are here suggested as possible gene-therapy pharmacodynamic biomarkers.