P4.28 Immunogenicity assay for detection of anti-dystrophin antibodies in serum of Duchenne Muscular Dystrophy patients following therapeutic antisense-induced exon skipping

Abstract

Antisense oligonucleotides (AONs) have been shown to induce exon skipping during dystrophin pre-mRNA splicing, and to result in novel dystrophin expression at muscle fiber membranes in Duchenne Muscular Dystrophy (DMD) patients. To monitor whether the novel dystrophin induces a humoral immune response, we developed a Western Blot screening assay to detect the presence of putative anti-dystrophin IgG antibodies in serum. A Western Blot membrane containing human dystrophin protein from healthy muscle protein lysate is hybridised with patient serum. Serum containing antibodies against dystrophin, cross react with the wild type human dystrophin protein on the Western Blot membrane and generate a band at the molecular weight corresponding to that of dystrophin. As part of qualification of this assay it was shown to detect a positive control mouse anti-human dystrophin antibody in serum from different healthy donors and in serum from DMD patients, demonstrating the specificity of the analytical method to detect the analyte (anti-human dystrophin antibody) in the presence of other components in the serum sample. The Western Blot assay was shown reproducibly to be sensitive enough to detect 250–500 ng of positive control anti-dystrophin antibody per ml of serum and thus is appropriate to support clinical studies. Furthermore, routine monitoring is possible, as serum samples in a trial can be easily obtained, stored (frozen) and assayed in batches. In the phase I/IIa clinical study in which 12 boys were treated with AON PRO051/GSK2402968 and dystrophin expression was observed in muscle fibers in post-treatment biopsies of 10/12 patients, this Western Blot assay was used and no anti-dystrophin antibodies were detected in the serum of the DMD treated boys.