Dystrophin and dystroglycan complex protein turnover dynamics after exon skipping therapy


Translational Research

Poster Number: Virtual


James Novak, PhD, Children's National Hospital; The George Washington University, Rita Spathis, Binghamton University, Utkarsh Dang, PhD, Binghamton University, Alyson Fiorillo, PhD, Children's National Hospital; The George Washington University, Ravi Hindupur, PhD, Children's National Hospital, Christopher Tully, MS, Children's Research Institute (CNMC), Davi Mazala, PhD, Towson University, Emily Canessa, Binghamton University, Kristy Brown, PhD, Solid Biosciences, Terence Partridge, PhD, Children's Research Institute (CNMC), Yetrib Hathout, PhD, Binghamton University, Kanneboyina Nagaraju, PhD, DVM, Binghamton University

Recently, the FDA granted accelerated approvals for four exon skipping therapies – Eteplirsen, Golodirsen, Viltolarsen, and Casimersen – for Duchenne Muscular Dystrophy (DMD). However, current exon skipping treatments have only demonstrated variable or sub-therapeutic levels of restored dystrophin protein in DMD patients, limiting their clinical impact. To understand variable protein expression and the behavior of restored truncated dystrophin protein in vivo, we assessed turnover dynamics of restored dystrophin and associated dystroglycan complex (DGC) proteins in mdx mice after exon skipping therapy, using a targeted, highly-reproducible and sensitive, in vivo stable isotope labeling mass spectrometry approach. Using this approach, we found that restored dystrophin protein exhibited altered stability and slower turnover in treated mdx muscles compared with that in wild type muscles. Assessment of mRNA transcript stability and dystrophin protein expression support our dystrophin protein turnover measurements and modeling. Further, we assessed pathology-induced muscle fiber turnover through bromodeoxyuridine labeling to model dystrophin and DGC protein turnover in the context of persistent muscle degeneration. Our findings reveal sequestration of restored dystrophin after exon skipping therapy in mdx muscle leading to a significant extension of its half-life compared to the dynamics of full-length dystrophin in normal muscle. In contrast, DGC proteins show constant, rapid turnover attributable to myofiber degeneration and dysregulation of the extracellular matrix in dystrophic muscle. Based on our results, we demonstrate the use of targeted mass spectrometry to evaluate the functionality of various restored dystrophin isoforms for DMD. As next generation exon skipping and gene therapies enter clinical development, we intend to utilize our approach to investigate the in vivo stability and functionality of various alternative gene therapy products to optimize their design, administration, and clinical use in DMD.