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.