361P Development of a next-generation multiplex ddPCR assay for measurement of in-frame dystrophin mRNA in people with DMD treated with BMN 351

Abstract

BMN 351, a next-generation antisense oligonucleotide (ASO), is being developed for the treatment of people with Duchenne muscular dystrophy (DMD) who have mutations amenable to exon 51 skipping. Out-of-frame dystrophin mRNA transcripts are converted by exon 51 skipping to in-frame, near-full–length, functional dystrophin transcripts. In-frame dystrophin mRNA expression is an early pharmacodynamic marker of BMN 351 activity. Current standard methods measure mRNA skipping by calculating the percentage of in-frame (exon skipped) dystrophin transcripts over total dystrophin transcripts. However, the relationship between exon skip percentage and dystrophin protein expression is unclear, as measurement of exon skip percentage does not account for reduced total dystrophin mRNA in DMD muscle. Here, we propose a novel assay strategy that quantifies functional in-frame dystrophin mRNA as a percentage of normal in-frame dystrophin transcripts from control muscle. This enhances the value of dystrophin mRNA measurements and better aligns with and predicts protein expression. Using in silico data mining and experimental verification, we identified both ubiquitously expressed and muscle-specific normalizer transcripts that can be measured in a multiplex ddPCR method with in-frame and total dystrophin mRNA. Ubiquitously expressed normalizers enable reporting of in-frame dystrophin mRNA levels normalized to total cellular content, compensating for varying RNA quantity and quality between samples. Muscle-specific normalizers provide insight into in-frame dystrophin mRNA expression per muscle cell in biopsies that may contain inflammatory infiltrate and adipocyte cells that vary with disease state and across individual biopsies. In this next-generation multiplex approach we propose a strategy to provide a more comprehensive and clinically relevant evaluation of mRNA expression that may be more predictive of functional dystrophin protein levels following ASO treatment for DMD.