Quantitation of Dystrophin and RGX-202 Microdystrophin by a Validated Hybrid LBA/LC-MS Assay


Clinical Trials

Poster Number: M154


Chen Wang, PhD, Regenxbio, Hiren Patel, PhD, REGENXBIO, Inc., Hua Wang, PhD, Regenxbio, Yanhong Wei, MD, MS, Regenxbio, Lin Yang, PhD, REGENXBIO, Inc., Michele Fiscella, PhD, REGENXBIO, Inc.

REGENXBIO’s RGX-202 AAV gene therapy program is designed to treat Duchenne Muscular Dystrophy (DMD) by delivering an optimized human microdystrophin transgene to DMD patients. A hybrid LBA/LC-MS/MS method was validated following the Guidance for Industry: Bioanalytical Method Validation (2018) to quantify the RGX-202 optimized microdystrophin as well as the full-length dystrophin in skeletal muscle tissues from human biopsies. A recombinant his-tagged RGX-202 microdystrophin protein, two signature peptides (one RGX-202 microdystrophin specific and another conserved in both RGX-202 microdystrophin and dystrophin), and two anti-peptide monoclonal antibodies were generated for the assay. Sections from the muscle tissue biopsies were homogenized, proteolyzed by trypsin, and the two signature peptides immunocaptured by anti-peptide antibodies pre-immobilized on magnetic beads, followed by acid elution and LC-MS/MS analysis.
The dynamic range was 25 – 2500 fmol per mg of total lysate protein with precision and accuracy less than 20% for both peptides. Full-length dystrophin levels in human biopsies ranged from ~ 1500 to 3300 fmol/mg across various muscle types. The LLOQ of RGX-202 microdystrophin was approximately 1.2% of mean dystrophin concentration in normal controls. This assay, as well as an orthogonal method that uses a Capillary Western Immunoassay (JESS) are currently used to support the first-in-human clinical study Affinity Duchenne. RGX-202 microdystrophin Data from the Affinity Duchenne study will be presented.