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 developed to quantify this optimized microdystrophin and full-length dystrophin in skeletal muscle tissues from mouse, monkey and human. Tissues were homogenized, proteolyzed by trypsin and followed by immunocapturing two peptides: one microdystrophin specific and another conserved in microdystrophin and dystrophin. A recombinant microdystrophin protein and two anti-peptide monoclonal antibodies were developed for the assay. The dynamic range was 12.5 – 2500 fmol per mg of lysate protein with precision and accuracy within 20% for both peptides. Full-length dystrophin levels in mouse, NHP and human were measured from 1000 to 3000 fmol/mg across various muscle types. The LLOQ of microdystrophin was approximately 0.5% of mean dystrophin concentration in normal controls. The assay will be validated to support the first-in-human clinical study.
Learning Objectives:
Upon completion, participant will be able to learn the progress of a novel AAV gene therapy approach for DMD disease.
Upon completion, participant will be able to understand the considerations behind choosing different approaches for tissue protein quantitation by various methods (LCMS, western blot, IHC and capillary WB).
Upon completion, participant will be able to understand development of an LCMS assay targeting analyte including a transgene product from AAV gene therapy and an endogenous protein.
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