The percentage of dystrophin positive fibers (PDPF) is a widely utilized analytical endpoint to measure efficacy of drug treatment in subjects receiving gene therapy for Duchenne muscular dystrophy (DMD). Although DMD is characterized by low or absent functional dystrophin expression, the complexity of the dystrophin-encoding gene allows expression of protein fragments that may or may not be functional. This is illustrated by applying both immunofluorescent labeling and mass spectrometry to quantify dystrophin content in non-dystrophic and dystrophic muscle biopsy samples. Epitopes associated with functional or non-functional dystrophin quantified by immunofluorescence in pre-treatment specimens may complicate the detection and interpretation of increases in dystrophin expression when the PDPF is used as an analytical endpoint to evaluate the treatment efficacy. In these situations, measurements of staining intensities, in addition to PDFP, may better discern the differential dystrophin expression between pre-treatment and post-treatment specimens. Thus, the H-score paradigm may improve the quantification (excellent analytical sensitivity and analytical specificity on a more exhaustive scale) of dystrophin expression by measuring both PDPF and the intensity of immunofluorescent staining.
To mitigate low measurement differentiation of PDPF, we have developed an image analysis-based H-scoring paradigm predicated on binning dystrophin expression measured by immunofluorescence analysis with multiple C-terminus and rod domain anti-dystrophin antibodies (e.g., DYS2, MANDYS106, and DYSB). This paradigm produces high H-score values in non-dystrophic, intermediate values in Becker muscular dystrophy (BMD), and generally low values in DMD specimens. In both DMD and BMD specimens, we detected a range of dystrophin expression, possibly related to the genetic heterogeneity of these groups. We found that such heterogeneity was observed more readily utilizing the H-score than the PDPF alone. Our data suggest the H-score approach provides more accurate quantification of dystrophin and related biomarkers, facilitating improved assessment of therapeutic agent efficacy and guiding drug development.